Methods and compositions for producing olefins

ABSTRACT

Compositions and methods for producing olefins are described herein. The olefins can be used to produced biofuels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of copending U.S. patent applicationSer. No. 12/673,752, filed Apr. 14, 2011, as the U.S. National phase ofPatent Cooperation Treaty Application No. PCT/US08/14029, filed Dec. 22,2008, which claims priority to U.S. Provisional Application No.61/092,278, filed on Aug. 27, 2008; U.S. Provisional Application No.61/051,886, filed on May 9, 2008; and U.S. Provisional Application No.61/016,183, filed on Dec. 21, 2007, all of which are herein expresslyincorporated by reference in their entirety.

INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety herein is a computer-readablenucleotide/amino acid sequence listing submitted concurrently herewithand identified as follows: One 40,960 Byte ASCII (Text) file named“LS00011 PCT_ST25_Sequence Lstg_(—)03.12.09” created on Mar. 11, 2009.It is understood that the Patent and Trademark Office will make thenecessary changes in application number and filing date for the instantapplication.

BACKGROUND OF THE INVENTION

Petroleum is a limited, natural resource found in the Earth in liquid,gaseous, or solid forms. Petroleum is primarily composed ofhydrocarbons, which are comprised mainly of carbon and hydrogen. It alsocontains significant amounts of other elements, such as, nitrogen,oxygen, or sulfur, in different forms.

Petroleum is a valuable resource, but petroleum products are developedat considerable costs, both financial and environmental. First, sourcesof petroleum must be discovered. Petroleum exploration is an expensiveand risky venture. The cost of exploring deep water wells can exceed$100 million. Moreover, there is no guarantee that these wells willcontain petroleum. It is estimated that only 40% of drilled wells leadto productive wells generating commercial hydrocarbons. In addition tothe economic cost, petroleum exploration carries a high environmentalcost. For example, offshore exploration disturbs the surrounding marineenvironments.

After a productive well is discovered, the petroleum must be extractedfrom the Earth at great expense. During primary recovery, the naturalpressure underground is sufficient to extract about 20% of the petroleumin the well. As this natural pressure falls, secondary recovery methodsare employed, if economical. Generally, secondary recovery involvesincreasing the well's pressure by, for example, water injection, naturalgas injection, or gas lift. Using secondary recovery methods, anadditional 5% to 15% of petroleum is recovered. Once secondary recoverymethods are exhausted, tertiary recovery methods can be used, ifeconomical. Tertiary methods involve reducing the viscosity of thepetroleum to make it easier to extract. Using tertiary recovery methods,an additional 5% to 15% of petroleum is recovered. Hence, even under thebest circumstances, only 50% of the petroleum in a well can beextracted. Petroleum extraction also carries an environmental cost. Forexample, petroleum extraction can result in large seepages of petroleumrising to the surface. Moreover, offshore drilling involves dredging theseabed which disrupts or destroys the surrounding marine environment.Since petroleum deposits are not found uniformly throughout the Earth,petroleum must be transported over great distances from petroleumproducing regions to petroleum consuming regions. In addition to theshipping costs, there is also the environmental risk of devastating oilspills.

In its natural form, crude petroleum extracted from the Earth has fewcommercial uses. It is a mixture of hydrocarbons (e.g., paraffins (oralkanes), olefins (or alkenes), alkynes, napthenes (or cylcoalkanes),aliphatic compounds, aromatic compounds, etc.) of varying length andcomplexity. In addition, crude petroleum contains other organiccompounds (e.g., organic compounds containing nitrogen, oxygen, sulfur,etc.) and impurities (e.g., sulfur, salt, acid, metals, etc.).

Hence, crude petroleum must be refined and purified before it can beused commercially. Due to its high energy density and its easytransportability, most petroleum is refined into fuels, such astransportation fuels (e.g., gasoline, diesel, aviation fuel, etc.),heating oil, liquefied petroleum gas, etc.

Crude petroleum is also a primary source of raw materials for producingpetrochemicals. The two main classes of raw materials derived frompetroleum are short chain olefins (e.g., ethylene and propylene) andaromatics (e.g., benzene and xylene isomers). These raw materials arederived from longer chain hydrocarbons in crude petroleum by cracking itat considerable expense using a variety of methods, such as catalyticcracking, steam cracking, or catalytic reforming. These raw materialsare used to make petrochemicals, which cannot be directly refined fromcrude petroleum, such as monomers, solvents, detergents, or adhesives.

One example of a raw material derived from crude petroleum is ethylene.Ethylene is used to produce petrochemicals such as, polyethylene,ethanol, ethylene oxide, ethylene glycol, polyester, glycol ether,ethoxylate, vinyl acetate, 1,2-dichloroethane, trichloroethylene,tetrachloroethylene, vinyl chloride, and polyvinyl chloride. Anadditional example of a raw material is propylene, which is used toproduce isopropyl alcohol, acrylonitrile, polypropylene, propyleneoxide, propylene glycol, glycol ethers, butylene, isobutylene,1,3-butadiene, synthetic elastomers, polyolefins, alpha-olefins, fattyalcohols, acrylic acid, acrylic polymers, allyl chloride,epichlorohydrin, and epoxy resins.

These petrochemicals can then be used to make specialty chemicals, suchas plastics, resins, fibers, elastomers, pharmaceuticals, lubricants, orgels. Particular specialty chemicals which can be produced frompetrochemical raw materials are: fatty acids, hydrocarbons (e.g., longchain, branched chain, saturated, unsaturated, etc.), fatty alcohols,esters, fatty aldehydes, ketones, lubricants, etc.

Specialty chemicals have many commercial uses. Hydrocarbons have manycommercial uses. For example, shorter chain alkanes are used as fuels.Methane and ethane are the main constituents of natural gas. Longerchain alkanes (e.g., from five to sixteen carbons) are used astransportation fuels (e.g., gasoline, diesel, or aviation fuel). Alkaneshaving more than sixteen carbon atoms are important components of fueloils and lubricating oils. Even longer alkanes, which are solid at roomtemperature, can be used, for example, as a paraffin wax. Alkanes thatcontain approximately thirty-five carbons are found in bitumen, which isused for road surfacing. In addition, longer chain alkanes can becracked to produce commercially useful shorter chain hydrocarbons.

Like short chain alkanes, short chain olefins, or alkenes, are used intransportation fuels. Longer chain olefins are used in plastics,lubricants, and synthetic lubricants. In addition, olefins are used as afeedstock to produce alcohols, esters, plasticizers, surfactants,tertiary amines, enhanced oil recovery agents, fatty acids, thiols,alkenylsuccinic anhydrides, polymers, textiles, solvents, adhesivesepoxides, chlorinated alkanes, chlorinated olefins, waxes, fueladditives, and drag flow reducers. In addition, long chain olefins canbe cracked to produce fuels.

Olefins have traditionally been produced from petroleum sources throughthe process of steam or catalytic cracking. Because of the limitedavailability and high cost of petroleum sources, the cost of producingolefins from such petroleum sources has been steadily increasing.

Olefins are the largest volume of chemical intermediates produced in thechemical industry, with global annual production previously estimated atover 300 billion lbs per year. Olefins are produced almost exclusivelyfrom ethane or other light alkanes (naphtha) in a process calledcracking. This process involves heating the ethane or other lightolefins to approximately 750-1000° C. in a cracker. It has beenestimated that 30% of all pollution from chemical plants comes fromcracking owing to emissions and unburned hydrocarbons in the flamerequired to heat the cracker. Approximately 10% of petroleum is consumedin the production of olefins and related chemicals.

In addition, crude petroleum is a source of lubricants. Lubricantsderived petroleum are typically composed of olefins, particularlypolyolefins and terminal olefins. Lubricants can either be refined fromcrude petroleum or manufactured using raw materials refined from crudepetroleum.

Obtaining these specialty chemicals from crude petroleum requires asignificant financial investment as well as a great deal of energy. Itis also an inefficient process because frequently the long chainhydrocarbons in crude petroleum are cracked to produce smaller monomers.These monomer are then used as the raw material to manufacture the morecomplex specialty chemicals.

In addition to the problems with exploring, extracting, transporting,and refining petroleum, petroleum is a limited and dwindling resource.One estimate of world petroleum consumption is 30 billion barrels peryear. By some estimates, it is predicted that at current productionlevels, the world's petroleum reserves could be depleted before the year2050.

Finally, the burning of petroleum based fuels releases greenhouse gases(e.g., carbon dioxide) and other forms of air pollution (e.g., carbonmonoxide, sulfur dioxide, etc.). As the world's demand for fuelincreases, the emission of greenhouse gases and other forms of airpollution also increases. The accumulation of greenhouse gases in theatmosphere leads to an increase global warming. Hence, in addition todamaging the environment locally (e.g., oil spills, dredging of marineenvironments, etc.), burning petroleum also damages the environmentglobally.

Due to the inherent challenges posed by petroleum, there is a need for arenewable petroleum source which does not need to be explored,extracted, transported over long distances, or substantially refinedlike petroleum. There is also a need for a renewable petroleum sourcewhich can be produced economically without creating the type ofenvironmental damage produced by the petroleum industry and the burningof petroleum based fuels. For similar reasons, there is also a need fora renewable source of chemicals which are typically derived frompetroleum.

One method of producing renewable petroleum is by engineeringmicroorganisms to produce renewable petroleum products. Somemicroorganims have a natural ability to produce chemicals. For example,yeast has been used for centuries to produce ethanol (e.g., beer, wine,etc.). In recent years, through the development of advancedbiotechnologies, it is possible to metabolically engineer an organism toproduce biochemicals that were never previously produced. Chemicalsderived from these cellular activities are known as biochemicals. Fuelsproduced these cellular activities are known as biofuels. Biofuels are arenewable alternative to petroleum based fuels. Biofuels can besubstituted for any petroleum based fuel (e.g., gasoline, diesel,aviation fuel, heating oil, etc.). Biofuels can be derived fromrenewable sources, such as plant matter, animal matter, or even wasteproducts. These renewable sources are collectively known as biomass. Oneadvantage of biofuels over petroleum based fuels is that they do notrequire expensive and risky exploration or extraction. In addition,biofuels can be locally produced. Hence, they do not requiretransportation over long distances. Moreover, biofuels can be madedirectly without the need for expensive and energy intensive refining asis needed with refining crude petroleum. In other circumstances, thebiofuel may require a limited and cost-effective level of refining.Furthermore, the use of biofuels improves the environment by reducingthe amount of environmentally harmful emissions (e.g., green housegases, air pollution, etc.) released during combustion. Finally,biofuels maintain a balanced carbon cycle because biofuels are producedfrom biomass, a renewable, natural resource. While the burning ofbiofuels will release carbon (e.g., as carbon dioxide), this carbon willbe recycled during the production of biomass (e.g., the cultivation ofcrops) thereby balancing the carbon cycle unlike petroleum based fuels.

For similar reasons, biologically derived chemicals offer the sameadvantages as biofuels over petroleum based fuels. Biologically derivedchemicals are a renewable alternative to petrochemicals. Biologicallyderived chemicals, such as hydrocarbons (e.g., alkanes, alkenes, oralkynes), fatty alcohols, esters, fatty acids, fatty aldehydes, andketones are superior to petrochemicals because they are produceddirectly without extensive refining. Unlike petrochemicals, biologicallyderived chemicals do not need to be refined like crude petroleum torecover raw materials which must then be further processed to make morecomplex petrochemicals. Biologically derived chemicals are directlyconverted from biomass to the desired chemical product.

To reduce our reliance on petroleum based fuels, it would be desirableto produce olefins from other sources.

SUMMARY OF THE INVENTION

The invention is based, at least in part, on the discovery of a novelgene, orf880, which encodes an olefin-producing enzyme. Accordingly, inone aspect, the invention features an isolated polynucleotide comprisinga nucleotide sequence having at least 70% sequence identity to SEQ IDNO:1, 4, 6, 8, 9, 11, or 13. In some embodiments, the nucleotidesequence has at least about 75%, at least about 80%, at least about 85%,at least about 90%, at least about 91%, at least about 92%, at leastabout 93%, at least about 94%, at least about 95%, at least about 96%,at least about 97%, at least about 98%, or at least about 99% sequenceidentity to SEQ ID NO:1, 4, 6, 8, 9, 11, or 13. In some embodiments, thepolynucleotide comprises the nucleotide sequence of SEQ ID NO:1, 4, 6,8, 9, 11, or 13. In other embodiments, the polynucleotide consists ofthe nucleotide sequence of SEQ ID NO:1, 4, 6, 8, 9, 11, or 13.

In another aspect, the invention features an isolated polynucleotidecomprising a nucleotide sequence that hybridizes to a complement of SEQID NO:1, 4, 6, 8, 9, 11, or 13 or to a fragment thereof. In someembodiments, the nucleotide sequence hybridizes to a complement of SEQID NO:1, 4, 6, 8, 9, 11, or 13 or to a fragment thereof under lowstringency, medium stringency, high stringency, or very high stringencyconditions.

In another aspect, the invention features an isolated polynucleotidecomprising a sequence encoding a polypeptide comprising (i) the aminoacid sequence of SEQ ID NO:2, 5, 7, 10, 12, or 14 or (ii) the amino acidsequence of SEQ ID NO:2, 5, 7, 10, 12, or 14 with one or more amino acidsubstitutions, additions, insertions, or deletions. In some embodiments,the polypeptide comprises the amino acid sequence of SEQ ID NO:2, 5, 7,10, 12, or 14 with one or more conservative amino acid substitutions.For example, the polypeptide comprises one or more of the followingconservative amino acid substitutions: replacement of an aliphatic aminoacid, such as alanine, valine, leucine, and isoleucine, with anotheraliphatic amino acid; replacement of a serine with a threonine;replacement of a threonine with a serine; replacement of an acidicresidue, such as aspartic acid and glutamic acid, with another acidicresidue; replacement of a residue bearing an amide group, such asasparagine and glutamine, with another residue bearing an amide group;exchange of a basic residue, such as lysine and arginine, with anotherbasic residue; and replacement of an aromatic residue, such asphenylalanine and tyrosine, with another aromatic residue. In someembodiments, the polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acidsubstitutions, additions, insertions, or deletions.

In some embodiments, the polypeptide has fatty acid decarboxylaseactivity.

In another aspect, the invention features an isolated polynucleotidecomprising a nucleotide sequence encoding a polypeptide having the samebiological activity as a polypeptide comprising the amino acid sequenceof SEQ ID NO:2, 5, 7, 10, 12, or 14, wherein the polynucleotidecomprises: (i) the nucleotide sequence of SEQ ID NO:1, 4, 6, 8, 9, 11,or 13 or a fragment thereof, or (ii) a nucleotide sequence thathybridizes to a complement of SEQ ID NO:1, 4, 6, 8, 9, 11, or 13 or to afragment thereof. In some embodiments, the biological activity is fattyacid decarboxylase activity. In some embodiments, the nucleotidesequence hybridizes to a complement of SEQ ID NO:1, 4, 6, 8, 9, 11, or13 or to a fragment thereof under low stringency, medium stringency,high stringency, or very high stringency conditions.

In some embodiments, the polynucleotide comprises a sequence encoding apolypeptide comprising the amino acid sequence of SEQ ID NO:2, 5, 7, 10,12, or 14.

In other embodiments, a polynucleotide described herein is isolated froma bacterium, plant, insect, yeast, fungus, or mammal. In someembodiments, the polynucleotide is isolated from a bacterium, forexample, a Gram positive or Gram negative bacterium. In specificembodiments, the bacterium is a member of the genus Jeotgalicoccus,Corynebacterium, Kokuria, Methylobacterium, or Bacillus. For example,the bacterium is selected from the group consisting of Jeotgalicoccushalotolerans, Jeotgalicoccus psychrophilus, Jeotgalicoccus sp. ATCC8456, Jeotgalicoccus pinnipedalis, Corynebacterium efficiens, Kokuriarhizophila, Methylobacterium populi, and Bacillus subtilis.

In other embodiments, the isolated polynucleotide includes an operablylinked promoter.

In another aspect, the invention features an isolated polypeptide thatincludes the amino acid sequence of SEQ ID NO:2, 5, 7, 10, 12, or 14, ora biologically active fragment thereof. In some embodiments, thepolypeptide consists of the amino acid sequence of SEQ ID NO:2, 5, 7,10, 12, or 14 or a biologically active fragment thereof. In someembodiments, the fragment is at least about 25 amino acids in length,for example, at least about 30, at least about 40, at least about 50, atleast about 75, at least about 100, at least about 150, at least about200, or at least about 250 amino acids in length. In certainembodiments, the fragment has fatty acid decarboxylase activity.

In yet another aspect, the invention features an isolated polypeptidethat includes the amino acid sequence of SEQ ID NO:2, 5, 7, 10, 12, or14 with one or more amino acid substitutions, additions, insertions, ordeletions. In some embodiments, the polypeptide includes the amino acidsequence of SEQ ID NO:2, 5, 7, 10, 12, or 14 with one or moreconservative amino acid substitutions. For example, the polypeptideincludes one or more of the following conservative amino acidsubstitutions: replacement of an aliphatic amino acid, such as alanine,valine, leucine, and isoleucine, with another aliphatic amino acid;replacement of a serine with a threonine; replacement of a threoninewith a serine; replacement of an acidic residue, such as aspartic acidand glutamic acid, with another acidic residue; replacement of a residuebearing an amide group, such as asparagine and glutamine, with anotherresidue bearing an amide group; exchange of a basic residue, such aslysine and arginine, with another basic residue; and replacement of anaromatic residue, such as phenylalanine and tyrosine, with anotheraromatic residue.

In some embodiments, the polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100 or more amino acidsubstitutions, additions, insertions, or deletions. In some embodiments,the isolated polypeptide has fatty acid decarboxylase activity.

In another aspect, the invention features an isolated polypeptide thatincludes the amino acid sequenceF-X-X-E-[VI]-[RK]-R-X-Y-P-F-{F}-P-X-[LIV], where X is any amino acid and{F} stands for any amino acid except Phe; and wherein the polypeptidehas fatty acid decarboxylase activity. In an alternate embodiment, theinvention features an isolated polypeptide that includes the amino acidsequence P-X(6)-[LI]-X(4)-{G}-[VI]-[HQM]-X-[MLV]-D-G-X(2)-H-X(2)-R-K,where X is any amino acid and {G} stands for any amino acid except Gly;and wherein the polypeptide has fatty acid decarboxylase activity. Inanother embodiment, the invention features an isolated polypeptide thatincludes the amino acid sequence[AC]-[AG]-[IV]-[DE]-[IL]-X-N-X(2)-R-P-X-[VI]-A-X(3)-[FY]-X(2)-F-X(3)-A-[LMV]-X-[DE]where X is any amino acid; and wherein the polypeptide has fatty aciddecarboxylase activity. In another embodiment, the invention features anisolated polypeptide that includes the amino acid sequence{RK}-X(6)-[LMV]-X(4)-[AC]-[AG]-[IV]-[DE]-[IL]-X-N-X(2)-R-P-X-[VI]-A-X(3)-[FY]-X(2)-F-X(3)-A-[LMV]-X-[DE]where X is any amino acid and {RK} stands for any amino acid except Argand Lys; and wherein the polypeptide has fatty acid decarboxylaseactivity.

In another aspect, the invention features a recombinant vector thatincludes a polynucleotide described herein. In some embodiments, thevector is a plasmid. In other embodiments, the vector further includes apromoter operably linked to the polynucleotide, for example, adevelopmentally-regulated, an organelle-specific, a tissue-specific, aninducible, a constitutive, or a cell-specific promoter.

In some embodiments, the vector includes at least one sequence selectedfrom the group consisting of: (a) a regulatory sequence operativelycoupled to the polynucleotide; (b) a selection marker operativelycoupled to the polynucleotide; (c) a marker sequence operatively coupledto the polynucleotide; (d) a purification moiety operatively coupled tothe polynucleotide; (e) a secretion sequence operatively coupled to thepolynucleotide; and (f) a targeting sequence operatively coupled to thepolynucleotide.

In another aspect, the invention features a host cell comprising arecombinant vector described herein, for example, a recombinant vectorthat includes a polynucleotide sequence described herein. In someembodiments, the host cell expresses a polypeptide encoded by therecombinant vector, for example, encoded by a polynucleotide sequenceincluded in the recombinant vector. In some embodiments, thepolynucleotide sequence is stably incorporated into the genomic DNA ofthe host cell. In particular embodiments, the expression of thepolynucleotide sequence is under the control of a regulated promoterregion.

In some embodiments, the host cell is a mammalian cell, plant cell,insect cell, yeast cell, fungus cell, a filamentous fungi cell, orbacterial cell.

In some embodiments, the host cell is a Gram positive bacterial cell. Inother embodiments, the host cell is a Gram negative bacterial host cell.

In some embodiments, the host cell is from a member of the genusEscherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas,Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor,Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete,Pleurotus, Trametes, Chrysosporium, Saccharomyces, Stenotrophomonas,Schizosaccharomyces, Yarrowia, or Streptomyces.

In particular embodiments, the host cell is a Bacillus lentus cell, aBacillus brevis cell, a Bacillus stearothermophilus cell, a Bacilluslicheniformis cell, a Bacillus alkalophilus cell, a Bacillus coagulanscell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillusthuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell,a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.

In other embodiments, the host cell is a Trichoderma koningii cell, aTrichoderma viride cell, a Trichoderma reesei cell, a Trichodermalongibrachiatum cell, an Aspergillus awamori cell, an Aspergillusfumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulanscell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicolainsolens cell, a Humicola lanuginose cell, a Rhodococcus opacus cell, aRhizomucor miehei cell, or a Mucor michei cell.

In yet other embodiments, the host cell is a Streptomyces lividans cellor a Streptomyces murinus cell. In other embodiments, the host cell isan Actinomycetes cell.

In some embodiments, the host cell is a CHO cell, a COS cell, a VEROcell, a BHK cell, a HeLa cell, a Cv1 cell, an MDCK cell, a 293 cell, a3T3 cell, or a PC12 cell.

In particular embodiments, the host cell is an E. coli cell, such as astrain B, a strain C, a strain K, or a strain W E. coli cell.

In certain embodiments, the host cell produces an olefin, for example, aterminal olefin. In some embodiments, the olefin is secreted by the hostcell.

In other embodiments, the host cell expresses a reduced level of anacyl-CoA synthase relative to a corresponding wild type host cell. Insome embodiments, a gene encoding an acyl-CoA synthase is attenuated ordeleted in the host cell. In certain embodiments, the gene is fadD,fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa1p, faa3p, lcfA, or the gene encoding the proteinhaving the amino acid sequence Accession No. ZP_(—)01644857. Inparticular embodiments, the gene is fadD.

In another aspect, the invention features a genetically engineeredmicroorganism comprising an exogenous control sequence stablyincorporated into the genomic DNA of the microorganism. In oneembodiment, the control sequence is integrated upstream of apolynucleotide comprising a nucleotide sequence having at least about70% sequence identity to SEQ ID NO:1, 4, 6, 8, 9, 11, or 13. In someembodiments, the nucleotide sequence has at least about 75%, at leastabout 80%, at least about 85%, at least about 90%, at least about 91%,at least about 92%, at least about 93%, at least about 94%, at leastabout 95%, at least about 96%, at least about 97%, at least about 98%,or at least about 99% sequence identity to SEQ ID NO:1, 4, 6, 8, 9, 11,or 13. In some embodiments, the nucleotide sequence is SEQ ID NO:1, 4,6, 8, 9, 11, or 13.

In some embodiments, the polynucleotide is endogenous to themicroorganism. In some embodiments, the microorganism expresses anincreased level of a polypeptide encoded by the polynucleotide relativeto a wild-type microorganism. In other embodiments, the microorganismproduces an increased level of an olefin relative to a wild-typemicroorganism. In some embodiments, the microorganism is a bacterium,such as a Jeotgalicoccus bacterium.

In other embodiments, the microorganism expresses a reduced level of anacyl-CoA synthase relative to a corresponding wild type microorganism.In some embodiments, a gene encoding an acyl-CoA synthase is attenuatedor deleted in the microorganism. In certain embodiments, the gene isfadD, fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa1p, faa3p, lcfA, or the gene encoding the proteinhaving the amino acid sequence Accession No. ZP_(—)01644857. Inparticular embodiments, the gene is fadD.

In another aspect, the invention features a polypeptide produced by ahost cell described herein.

In yet another aspect, the invention features a method of producing anolefin. The method comprises culturing a host cell described herein, forexample, a host cell comprising a nucleotide sequence described herein,under conditions sufficient to allow expression of a polypeptide encodedby the nucleotide sequence.

In some embodiments, the nucleotide sequence has at least about 70%, atleast about 75%, at least about 80%, at least about 85%, at least about90%, at least about 91%, at least about 92%, at least about 93%, atleast about 94%, at least about 95%, at least about 96%, at least about97%, at least about 98%, or at least about 99% sequence identity to SEQID NO:1, 4, 6, 8, 9, 11, or 13. In some embodiments, the nucleotidesequence is SEQ ID NO:1, 4, 6, 8, 9, 11, or 13.

In other embodiments, the nucleotide sequence hybridizes to a complementof SEQ ID NO:1, 4, 6, 8, 9, 11, or 13 or to a fragment thereof, forexample, under low stringency, medium stringency, high stringency, orvery high stringency conditions.

In other embodiments, the nucleotide sequence encodes a polypeptidecomprising: (i) the amino acid sequence of SEQ ID NO:2, 5, 7, 10, 12, or14; or (ii) the amino acid sequence of SEQ ID NO:2, 5, 7, 10, 12, or 14with one or more amino acid substitutions, additions, insertions, ordeletions. In some embodiments, the polypeptide comprises the amino acidsequence of SEQ ID NO:2, 5, 7, 10, 12, or 14 with one or moreconservative amino acid substitutions. For example, the polypeptidecomprises one or more of the following conservative amino acidsubstitutions: replacement of an aliphatic amino acid, such as alanine,valine, leucine, and isoleucine, with another aliphatic amino acid;replacement of a serine with a threonine; replacement of a threoninewith a serine; replacement of an acidic residue, such as aspartic acidand glutamic acid, with another acidic residue; replacement of a residuebearing an amide group, such as asparagine and glutamine, with anotherresidue bearing an amide group; exchange of a basic residue, such aslysine and arginine, with another basic residue; and replacement of anaromatic residue, such as phenylalanine and tyrosine, with anotheraromatic residue. In some embodiments, the polypeptide has about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 100, ormore amino acid substitutions, additions, insertions, or deletions. Insome embodiments, the polypeptide has fatty acid decarboxylase activity.

In other embodiments, the nucleotide sequence encodes a polypeptidehaving the same biological activity as a polypeptide comprising theamino acid sequence of SEQ ID NO:2, 5, 7, 10, 12, or 14. In someembodiments, the nucleotide sequence is SEQ ID NO:1, 4, 6, 8, 9, 11, or13 or a fragment thereof. In other embodiments, the nucleotide sequencehybridizes to a complement of SEQ ID NO:1, 4, 6, 8, 9, 11, or 13 or to afragment thereof, for example, under low stringency, medium stringency,high stringency, or very high stringency conditions. In someembodiments, the biological activity is fatty acid decarboxylaseactivity.

In some embodiments, the nucleotide sequence encodes a polypeptidecomprising the amino acid sequence of SEQ ID NO:2, 5, 7, 10, 12, or 14.

In some embodiments, the host cell is selected from the group consistingof a mammalian cell, plant cell, insect cell, yeast cell, fungus cell,filamentous fungi cell, and bacterial cell.

In some embodiments, the host cell is a Gram positive bacterial cell. Inother embodiments, the host cell is a Gram negative bacterial cell.

In some embodiments, the host cell is selected from the genusEscherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas,Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor,Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete,Pleurotus, Trametes, Chrysosporium, Saccharomyces, and Stenotrophamonas,

In particular embodiments, the host cell is a Bacillus lentus cell, aBacillus brevis cell, a Bacillus stearothermophilus cell, a Bacilluslicheniformis cell, a Bacillus alkalophilus cell, a Bacillus coagulanscell, a Bacillus circulans cell, a Bacillus pumilis cell, a Bacillusthuringiensis cell, a Bacillus clausii cell, a Bacillus megaterium cell,a Bacillus subtilis cell, or a Bacillus amyloliquefaciens cell.

In other embodiments, the host cell is a Trichoderma koningii cell, aTrichoderma viride cell, a Trichoderma reesei cell, a Trichodermalongibrachiatum cell, an Aspergillus awamori cell, an Aspergillusfumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulanscell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicolainsolens cell, a Humicola lanuginose cell, a Rhodococcus opacus cell, aRhizomucor miehei cell, or a Mucor michei cell.

In yet other embodiments, the host cell is a Streptomyces lividans cellor a Streptomyces murinus cell. In other embodiments, the host cell isan Actinomycetes cell.

In some embodiments, the host cell is a CHO cell, a COS cell, a VEROcell, a BHK cell, a HeLa cell, a Cv1 cell, an MDCK cell, a 293 cell, a3T3 cell, or a PC12 cell.

In particular embodiments, the host cell is an E. coli cell, such as astrain B, a strain C, a strain K, or a strain W E. coli cell.

In some embodiments, the olefin is a terminal olefin. In someembodiments, the terminal olefin is a C₃-C₂₅ terminal olefin. Forexample, the terminal olefin is a C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, or C₂₅terminal olefin. In some embodiments, the terminal olefin is nonadecene,methylnonadecene, heptadecene, methylheptadecene, or pentadecene.

In some embodiments, the terminal olefin is an unsaturated terminalolefin or a monounsaturated terminal olefin. In yet other embodiments,the terminal olefin is a straight chain terminal olefin, a branchedchain terminal olefin, or a terminal olefin that includes a cyclicmoiety.

In certain embodiments, the host cell overexpresses a fatty acidsubstrate. In some embodiments, the method further includes transformingthe host cell with a nucleic acid that encodes a fatty acid enzyme(e.g., a fatty acid enzyme described herein) and the host celloverexpresses a fatty acid substrate. In other embodiments, the methodfurther includes culturing the host cell in the presence of at least onefatty acid substrate.

In certain embodiments, the fatty acid substrate is a C₆-C₂₆ fatty acidsubstrate. For example, the fatty acid substrate is a C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃,C₂₄, C₂₅, or a C₂₆ fatty acid substrate. In particular embodiments, thefatty acid substrate is methyl eicosanoic acid, eicosanoic acid, methyloctadecanoic acid, stearic acid, or palmitic acid.

In some embodiments, the fatty acid substrate is an unsaturated fattyacid substrate, a monounsaturated fatty acid substrate, or a saturatedfatty acid substrate. In other embodiments, the fatty acid substrate isa straight chain fatty acid substrate, a branched chain fatty acidsubstrate, or a fatty acid substrate that includes a cyclic moiety.

In other embodiments, the host cell expresses a reduced level of anacyl-CoA synthase relative to a corresponding wild type host cell. Insome embodiments, a gene encoding an acyl-CoA synthase is attenuated ordeleted in the host cell. In certain embodiments, the gene is fadD,fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa1p, faa3p, lcfA, or the gene encoding the proteinhaving the amino acid sequence Accession No. ZP_(—)01644857. Inparticular embodiments, the gene is fadD.

In some embodiments, the method further includes isolating the olefinfrom the host cell or from the culture medium. In other embodiments, themethod further includes cracking or refining the olefin.

In another aspect, the invention features a method of making an olefin.The method includes contacting a biological substrate with an isolatedpolypeptide having the same biological activity as a polypeptidecomprising the amino acid sequence of SEQ ID NO:2, 5, 7, 10, 12, or 14.

In some embodiments, the olefin is a terminal olefin. In someembodiments, the terminal olefin is a C₃-C₂₅ terminal olefin. Forexample, the terminal olefin is a C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, or C₂₅terminal olefin. In some embodiments, the terminal olefin is nonadecene,methylnonadecene, heptadecene, methylheptadecene, or pentadecene.

In some embodiments, the terminal olefin is an unsaturated terminalolefin or a monounsaturated terminal olefin. In yet other embodiments,the terminal olefin is a straight chain terminal olefin, a branchedchain terminal olefin, or a terminal olefin that includes a cyclicmoiety.

In some embodiments, the biological substrate is a fatty acid substrate.In certain embodiments, the fatty acid substrate is a C₆-C₂₆ fatty acidsubstrate. For example, the fatty acid substrate is a C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃,C₂₄, C₂₅, or a C₂₆ fatty acid substrate. In particular embodiments, thefatty acid substrate is methyl eicosanoic acid, eicosanoic acid, methyloctadecanoic acid, stearic acid, or palmitic acid.

In some embodiments, the fatty acid substrate is an unsaturated fattyacid substrate, a monounsaturated fatty acid substrate, or a saturatedfatty acid substrate. In other embodiments, the fatty acid substrate isa straight chain fatty acid substrate, a branched chain fatty acidsubstrate, or a fatty acid substrate that includes a cyclic moiety.

In another aspect, the invention features a method of making an olefin.The method includes contacting a biological substrate with an isolatedpolypeptide encoded by a nucleotide sequence described herein. In someembodiments, the nucleotide sequence has at least about 70%, at leastabout 75%, at least about 80%, at least about 85%, at least about 90%,at least about 91%, at least about 92%, at least about 93%, at leastabout 94%, at least about 95%, at least about 96%, at least about 97%,at least about 98%, or at least about 99% sequence identity to SEQ IDNO:1, 4, 6, 8, 9, 11, or 13. In some embodiments, the nucleotidesequence is SEQ ID NO:1, 4, 6, 8, 9, 11, or 13.

In other embodiments, the nucleotide sequence hybridizes to a complementof SEQ ID NO:1, 4, 6, 8, 9, 11, or 13 or to a fragment thereof, forexample, under low stringency, medium stringency, high stringency, orvery high stringency conditions.

In some embodiments, the olefin is a terminal olefin. In someembodiments, the terminal olefin is a C₃-C₂₅ terminal olefin. Forexample, the terminal olefin is a C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, or C₂₅terminal olefin. In some embodiments, the terminal olefin is nonadecene,methylnonadecene, heptadecene, methylheptadecene, or pentadecene.

In some embodiments, the terminal olefin is an unsaturated terminalolefin or a monounsaturated terminal olefin. In yet other embodiments,the terminal olefin is a straight chain terminal olefin, a branchedchain terminal olefin, or a terminal olefin that includes a cyclicmoiety.

In some embodiments, the biological substrate is a fatty acid substrate.In certain embodiments, the fatty acid substrate is a C₆-C₂₆ fatty acidsubstrate. For example, the fatty acid substrate is a C₆, C₇, C₈, C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃,C₂₄, C₂₅, or a C₂₆ fatty acid substrate. In particular embodiments, thefatty acid substrate is methyl eicosanoic acid, eicosanoic acid, methyloctadecanoic acid, stearic acid, or palmitic acid.

In some embodiments, the fatty acid substrate is an unsaturated fattyacid substrate, a monounsaturated fatty acid substrate, or a saturatedfatty acid substrate. In other embodiments, the fatty acid substrate isa straight chain fatty acid substrate, a branched chain fatty acidsubstrate, or a fatty acid substrate that includes a cyclic moiety.

In another aspect, the invention features a method of making an olefin.The method includes contacting a biological substrate with an isolatedpolypeptide described herein. In some embodiments, the polypeptideincludes the amino acid sequence of SEQ ID NO:2, 5, 7, 10, 12, or 14 ora biologically active fragment thereof. In some embodiments, thepolypeptide consists of the amino acid sequence of SEQ ID NO:2, 5, 7,10, 12, or 14 or a biologically active fragment thereof. In someembodiments, the fragment is at least about 25 amino acids in length,for example, at least about 30, at least about 40, at least about 50, atleast about 75, at least about 100, at least about 150, at least about200, or at least about 250 amino acids in length. In certainembodiments, the fragment has fatty acid decarboxylase activity.

In other embodiments, the polypeptide includes the amino acid sequenceof SEQ ID NO:2, 5, 7, 10, 12, or 14 with one or more amino acidsubstitutions, additions, insertions, or deletions. In some embodiments,the polypeptide includes the amino acid sequence of SEQ ID NO:2, 5, 7,10, 12, or 14 with one or more conservative amino acid substitutions.For example, the polypeptide includes one or more of the followingconservative amino acid substitutions: replacement of an aliphatic aminoacid, such as alanine, valine, leucine, and isoleucine, with anotheraliphatic amino acid; replacement of a serine with a threonine;replacement of a threonine with a serine; replacement of an acidicresidue, such as aspartic acid and glutamic acid, with another acidicresidue; replacement of a residue bearing an amide group, such asasparagine and glutamine, with another residue bearing an amide group;exchange of a basic residue, such as lysine and arginine, with anotherbasic residue; and replacement of an aromatic residue, such asphenylalanine and tyrosine, with another aromatic residue. In someembodiments, the polypeptide has about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,15, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more amino acidsubstitutions, additions, insertions, or deletions. In some embodiments,the isolated polypeptide has fatty acid decarboxylase activity.

In other embodiments, the polypeptide includes the amino acid sequenceF-X-X-E-[VI]-[RK]-R-X-Y-P-F-{F}-P-X-[LIV]; and wherein the polypeptidehas fatty acid decarboxylase activity. In an alternate embodiment, thepolypeptide includes the amino acid sequenceP-X(6)-[LI]-X(4)-{G}-[VI]-[HQM]-X-[MLV]-D-G-X(2)-H-X(2)-R-K; and whereinthe polypeptide has fatty acid decarboxylase activity. In anotherembodiment, the polypeptide includes the amino acid sequence[AC]-[AG]-[IV]-[DE]-[IL]-X-N-X(2)-R-P-X-[VI]-A-X(3)-[FY]-X(2)-F-X(3)-A-[LMV]-X-[DE];and wherein the polypeptide has fatty acid decarboxylase activity. Inanother embodiment, the polypeptide includes the amino acid sequence{RK}-X(6)-[LMV]-X(4)-[AC]-[AG]-[IV]-[DE]-[IL]-X-N-X(2)-R-P-X-[VI]-A-X(3)-[FY]-X(2)-F-X(3)-A-[LMV]-X-[DE];and wherein the polypeptide has fatty acid decarboxylase activity. Inthe sequences described herein, X is any amino acid, [AB] is A or B(e.g., [VI] is valine or isoleucine), {A} is any amino acid except A(e.g., {F} stands for any amino acid except Phe), X is any amino acid,and X(n) is X repeated n times (e.g., X(4) is X-X-X-X).

In some embodiments, the olefin is a terminal olefin. In someembodiments, the terminal olefin is a C₃-C₂₅ terminal olefin. Forexample, the terminal olefin is a C₃, C₄, C₅, C₆, C₇, C₈, C₉, C₁₀, C₁₁,C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃, C₂₄, or C₂₅terminal olefin. In some embodiments, the terminal olefin is nonadecene,methylnonadecene, heptadecene, methylheptadecene, or pentadecene.

In some embodiments, the terminal olefin is an unsaturated terminalolefin or a monounsaturated terminal olefin. In yet other embodiments,the terminal olefin is a straight chain terminal olefin, a branchedchain terminal olefin, or a terminal olefin that includes a cyclic

In some embodiments, the biological substrate is a fatty acid substrate.In certain embodiments, the fatty acid substrate is a C₆-C₂₆ fatty acidsubstrate. For example, the fatty acid substrate is a C₆, C₇, C_(g), C₉,C₁₀, C₁₁, C₁₂, C₁₃, C₁₄, C₁₅, C₁₆, C₁₇, C₁₈, C₁₉, C₂₀, C₂₁, C₂₂, C₂₃,C₂₄, C₂₅, or a C₂₆ fatty acid substrate. In particular embodiments, thefatty acid substrate is methyl eicosanoic acid, eicosanoic acid, methyloctadecanoic acid, stearic acid, or palmitic acid.

In some embodiments, the fatty acid substrate is an unsaturated fattyacid substrate, a monounsaturated fatty acid substrate, or a saturatedfatty acid substrate. In other embodiments, the fatty acid substrate isa straight chain fatty acid substrate, a branched chain fatty acidsubstrate, or a fatty acid substrate that includes a cyclic moiety.

In another aspect, the invention features an olefin produced by any ofthe methods described herein. In particular embodiments, the olefin hasa δ¹³C of about −15.4 or greater. For example, the olefin has a δ¹³C ofabout −15.4 to about −10.9, for example, about −13.92 to about −13.84.In other embodiments, the olefin has an f_(M) ¹⁴C of at least about1.003. For example, the olefin has an f_(M) ¹⁴C of at least about 1.01or at least about 1.5. In some embodiments, the olefin has an f_(M) ¹⁴Cof about 1.111 to about 1.124.

In another aspect, the invention features a biofuel that includes anolefin produced by any of the methods described herein. In particularembodiments, the olefin has a δ¹³C of about −15.4 or greater. Forexample, the olefin has a δ¹³C of about −15.4 to about −10.9, forexample, about −13.92 to about −13.84. In other embodiments, the olefinhas an f_(M) ¹⁴C of at least about 1.003. For example, the olefin has anf_(M) ¹⁴C of at least about 1.01 or at least about 1.5. In someembodiments, the olefin has an f_(M) ¹⁴C of about 1.111 to about 1.124.In some embodiments, the biofuel is a biodiesel, gasoline, or jet fuel.

DEFINITIONS

Throughout the specification, a reference may be made using anabbreviated gene name or polypeptide name, but it is understood thatsuch an abbreviated gene or polypeptide name represents the genus ofgenes or polypeptides. Such gene names include all genes encoding thesame polypeptide and homologous polypeptides having the samephysiological function. Polypeptide names include all polypeptides thathave the same activity (e.g., that catalyze the same fundamentalchemical reaction).

The accession numbers referenced herein are derived from the NCBIdatabase (National Center for Biotechnology Information) maintained bythe National Institute of Health, U.S.A. The accession numbers are asprovided in the database as of Aug. 28, 2008.

EC numbers are established by the Nomenclature Committee of theInternational Union of Biochemistry and Molecular Biology (NC-IUBMB).The EC numbers referenced herein are derived from the KEGG Liganddatabase, maintained by the Kyoto Encyclopedia of Genes and Genomics,sponsored in part by the University of Tokyo. The EC numbers are asprovided in the database as of March, 2008.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

The term “about” is used herein to mean a value±20% of a given numericalvalue. Thus, “about 60%” means a value of between 60±(20% of 60) (i.e.,between 48 and 70).

As used herein, the term “attenuate” means to weaken, reduce ordiminish. For example, a polypeptide can be attenuated by modifying thepolypeptide to reduce its activity (e.g., by modifying a nucleotidesequence that encodes the polypeptide).

As used herein, the term “biodiesel” means a biofuel that can be asubstitute of diesel, which is derived from petroleum. Biodiesel can beused in internal combustion diesel engines in either a pure form, whichis referred to as “neat” biodiesel, or as a mixture in any concentrationwith petroleum-based diesel. Biodiesel can include esters orhydrocarbons, such as olefins (e.g., terminal olefins).

As used therein, the term “biofuel” refers to any fuel derived frombiomass. Biofuels can be substituted for petroleum based fuels. Forexample, biofuels are inclusive of transportation fuels (e.g., gasoline,diesel, jet fuel, etc.), heating fuels, and electricity-generatingfuels. Biofuels are a renewable energy source.

As used herein, the term “biomass” refers to a carbon source derivedfrom biological material. Biomass can be converted into a biofuel. Oneexemplary source of biomass is plant matter. For example, corn, sugarcane, or switchgrass can be used as biomass. Another non-limitingexample of biomass is animal matter, for example cow manure. Biomassalso includes waste products from industry, agriculture, forestry, andhouseholds. Examples of such waste products that can be used as biomassare fermentation waste, straw, lumber, sewage, garbage, and foodleftovers. Biomass also includes sources of carbon, such ascarbohydrates (e.g., monosaccharides, disaccharides, orpolysaccharides).

As used herein, the phrase “carbon source” refers to a substrate orcompound suitable to be used as a source of carbon for prokaryotic orsimple eukaryotic cell growth. Carbon sources can be in various forms,including, but not limited to polymers, carbohydrates, acids, alcohols,aldehydes, ketones, amino acids, peptides, and gases (e.g., CO and CO₂).These include, for example, various monosaccharides, such as glucose,fructose, mannose, and galactose; oligosaccharides, such asfructo-oligosaccharide and galacto-oligosaccharide; polysaccharides suchas xylose and arabinose; disaccharides, such as sucrose, maltose, andturanose; cellulosic material, such as methyl cellulose and sodiumcarboxymethyl cellulose; saturated or unsaturated fatty acid esters,such as succinate, lactate, and acetate; alcohols, such as ethanol ormixtures thereof. The carbon source can also be a product ofphotosynthesis, including, but not limited to, glucose. A preferredcarbon source is biomass. Another preferred carbon source is glucose.

As used herein, a “cloud point lowering additive” is an additive addedto a composition to decrease or lower the cloud point of a solution.

As used herein, the phrase “cloud point of a fluid” means thetemperature at which dissolved solids are no longer completely soluble.Below this temperature, solids begin precipitating as a second phasegiving the fluid a cloudy appearance. In the petroleum industry, cloudpoint refers to the temperature below which a solidified material orother heavy hydrocarbon crystallizes in a crude oil, refined oil, orfuel to form a cloudy appearance. The presence of solidified materialsinfluences the flowing behavior of the fluid, the tendency of the fluidto clog fuel filters, injectors, etc., the accumulation of solidifiedmaterials on cold surfaces (e.g., a pipeline or heat exchanger fouling),and the emulsion characteristics of the fluid with water.

A nucleotide sequence is “complementary” to another nucleotide sequenceif each of the bases of the two sequences matches (i.e., is capable offorming Watson Crick base pairs). The term “complementary strand” isused herein interchangeably with the term “complement”. The complementof a nucleic acid strand can be the complement of a coding strand or thecomplement of a non-coding strand.

As used herein, the term “conditions sufficient to allow expression”means any conditions that allow a host cell to produce a desiredproduct, such as a polypeptide or olefin described herein. Suitableconditions include, for example, fermentation conditions. Fermentationconditions can comprise many parameters, such as temperature ranges,levels of aeration, and media composition. Each of these conditions,individually and in combination, allows the host cell to grow. Exemplaryculture media include broths or gels. Generally, the medium includes acarbon source, such as glucose, fructose, cellulose, or the like, thatcan be metabolized by a host cell directly. In addition, enzymes can beused in the medium to facilitate the mobilization (e.g., thedepolymerization of starch or cellulose to fermentable sugars) andsubsequent metabolism of the carbon source.

To determine if conditions are sufficient to allow expression, a hostcell can be cultured, for example, for about 4, 8, 12, 24, 36, or 48hours. During and/or after culturing, samples can be obtained andanalyzed to determine if the conditions allow expression. For example,the host cells in the sample or the medium in which the host cells weregrown can be tested for the presence of a desired product. When testingfor the presence of a product, assays, such as, but not limited to, TLC,HPLC, GC/FID, GC/MS, LC/MS, MS, can be used.

It is understood that the polypeptides described herein may haveadditional conservative or non-essential amino acid substitutions, whichdo not have a substantial effect on the polypeptide functions. Whetheror not a particular substitution will be tolerated (i.e., will notadversely affect desired biological properties, such as decarboxylaseactivity) can be determined as described in Bowie et al., Science (1990)247:1306-1310. A “conservative amino acid substitution” is one in whichthe amino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include amino acidswith basic side chains (e.g., lysine, arginine, histidine), acidic sidechains (e.g., aspartic acid, glutamic acid), uncharged polar side chains(e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine,cysteine), nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan),beta-branched side chains (e.g., threonine, valine, isoleucine), andaromatic side chains (e.g., tyrosine, phenylalanine, tryptophan,histidine).

As used herein, “control element” means a transcriptional controlelement. Control elements include promoters and enhancers. The term“promoter element,” “promoter,” or “promoter sequence” refers to a DNAsequence that functions as a switch that activates the expression of agene. If the gene is activated, it is said to be transcribed orparticipating in transcription. Transcription involves the synthesis ofmRNA from the gene. A promoter, therefore, serves as a transcriptionalregulatory element and also provides a site for initiation oftranscription of the gene into mRNA. Control elements interactspecifically with cellular proteins involved in transcription (Maniatiset al., Science 236:1237, 1987).

As used herein, the term “fatty acid” means a carboxylic acid having theformula RCOOH. R represents an aliphatic group, preferably an alkylgroup. R can comprise between about 4 and about 22 carbon atoms. Fattyacids can be saturated, monounsaturated, or polyunsaturated. In apreferred embodiment, the fatty acid is made from a fatty acidbiosynthetic pathway.

As used herein, the term “fatty acid biosynthetic pathway” means abiosynthetic pathway that produces fatty acids. The fatty acidbiosynthetic pathway includes fatty acid enzymes that can be engineered,as described herein, to produce fatty acids, and in some embodiments canbe expressed with additional enzymes to produce fatty acids havingdesired carbon chain characteristics.

As used herein, “fatty acid enzyme” means any enzyme involved in fattyacid biosynthesis. Fatty acid enzymes can be expressed or overexpressedin host cells to produce fatty acids. Non-limiting examples of fattyacid enzymes include fatty acid synthases and thioesterases.

As used herein, “fraction of modern carbon” or “f_(M)” has the samemeaning as defined by National Institute of Standards and Technology(NIST) Standard Reference Materials (SRMs) 4990B and 4990C, known asoxalic acids standards HOxI and HOxII, respectively. The fundamentaldefinition relates to 0.95 times the ¹⁴C/¹²C isotope ratio HOxI(referenced to AD 1950). This is roughly equivalent to decay-correctedpre-Industrial Revolution wood. For the current living biosphere (plantmaterial), f_(M) is approximately 1.1.

Calculations of “homology” between two sequences can be performed asfollows. The sequences are aligned for optimal comparison purposes(e.g., gaps can be introduced in one or both of a first and a secondamino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, the length of a reference sequence that isaligned for comparison purposes is at least about 30%, preferably atleast about 40%, more preferably at least about 50%, even morepreferably at least about 60%, and even more preferably at least about70%, at least about 80%, at least about 90%, or about 100% of the lengthof the reference sequence. The amino acid residues or nucleotides atcorresponding amino acid positions or nucleotide positions are thencompared. When a position in the first sequence is occupied by the sameamino acid residue or nucleotide as the corresponding position in thesecond sequence, then the molecules are identical at that position (asused herein, amino acid or nucleic acid “identity” is equivalent toamino acid or nucleic acid “homology”). The percent identity between thetwo sequences is a function of the number of identical positions sharedby the sequences, taking into account the number of gaps and the lengthof each gap, which need to be introduced for optimal alignment of thetwo sequences.

The comparison of sequences and determination of percent homologybetween two sequences can be accomplished using a mathematicalalgorithm. In a preferred embodiment, the percent homology between twoamino acid sequences is determined using the Needleman and Wunsch(1970), J. Mol. Biol. 48:444-453, algorithm that has been incorporatedinto the GAP program in the GCG software package, using either a Blossum62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6,or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet anotherpreferred embodiment, the percent homology between two nucleotidesequences is determined using the GAP program in the GCG softwarepackage, using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60,70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularlypreferred set of parameters (and the one that should be used if thepractitioner is uncertain about which parameters should be applied todetermine if a molecule is within a homology limitation of the claims)are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extendpenalty of 4, and a frameshift gap penalty of 5.

As used herein, a “host cell” is a cell used to produce a productdescribed herein (e.g., an olefin described herein). A host cell can bemodified to express or overexpress selected genes or to have attenuatedexpression of selected genes. Non-limiting examples of host cellsinclude plant, animal, human, bacteria, yeast, or filamentous fungicells.

As used herein, the term “hybridizes under low stringency, mediumstringency, high stringency, or very high stringency conditions”describes conditions for hybridization and washing. Guidance forperforming hybridization reactions can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. Aqueousand nonaqueous methods are described in that reference and either methodcan be used. Specific hybridization conditions referred to herein are asfollows: 1) low stringency hybridization conditions in 6× sodiumchloride/sodium citrate (SSC) at about 45° C., followed by two washes in0.2×SSC, 0.1% SDS at least at 50° C. (the temperature of the washes canbe increased to 55° C. for low stringency conditions); 2) mediumstringency hybridization conditions in 6×SSC at about 45° C., followedby one or more washes in 0.2×SSC, 0.1% SDS at 60° C.; 3) high stringencyhybridization conditions in 6×SSC at about 45° C., followed by one ormore washes in 0.2×SSC, 0.1% SDS at 65° C.; and preferably 4) very highstringency hybridization conditions are 0.5M sodium phosphate, 7% SDS at65° C., followed by one or more washes at 0.2×SSC, 1% SDS at 65° C. Veryhigh stringency conditions (4) are the preferred conditions unlessotherwise specified.

The term “isolated” as used herein with respect to nucleic acids, suchas DNA or RNA, refers to molecules separated from other DNAs or RNAs,respectively, that are present in the natural source of the nucleicacid. Moreover, by an “isolated nucleic acid” is meant to includenucleic acid fragments, which are not naturally occurring as fragmentsand would not be found in the natural state. The term “isolated” is alsoused herein to refer to polypeptides, which are isolated from othercellular proteins and is meant to encompass both purified andrecombinant polypeptides. The term “isolated” as used herein also refersto a nucleic acid or peptide that is substantially free of cellularmaterial, viral material, or culture medium when produced by recombinantDNA techniques. The term “isolated” as used herein also refers to anucleic acid or peptide that is substantially free of chemicalprecursors or other chemicals when chemically synthesized.

As used herein, the “level of expression of a gene in a cell” refers tothe level of mRNA, pre-mRNA nascent transcript(s), transcript processingintermediates, mature mRNA(s), and degradation products encoded by thegene in the cell.

As used herein, the term “microorganism” means prokaryotic andeukaryotic microbial species from the domains Archaea, Bacteria andEucarya, the latter including yeast and filamentous fungi, protozoa,algae, or higher Protista. The terms “microbial cells” (i.e., cells frommicrobes) and “microbes” are used interchangeably and refer to cells orsmall organisms that can only be seen with the aid of a microscope.

As used herein, the term “nucleic acid” refers to polynucleotides, suchas deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid(RNA). The term should also be understood to include, as equivalents,analogs of either RNA or DNA made from nucleotide analogs, and, asapplicable to the embodiment being described, single (sense orantisense) and double-stranded polynucleotides, ESTs, chromosomes,cDNAs, mRNAs, and rRNAs.

“Olefin” and “alkene” are used interchangeably herein, and “terminalolefin,” “α-olefin,” and “terminal alkene” are used interchangeablyherein.

As used herein, the term “operably linked” means that selectednucleotide sequence (e.g., encoding a polypeptide described herein) isin proximity with a promoter to allow the promoter to regulateexpression of the selected DNA. In addition, the promoter is locatedupstream of the selected nucleotide sequence in terms of the directionof transcription and translation. By “operably linked” is meant that anucleotide sequence and a regulatory sequence(s) are connected in such away as to permit gene expression when the appropriate molecules (e.g.,transcriptional activator proteins) are bound to the regulatorysequence(s).

The term “or” is used herein to mean, and is used interchangeably with,the term “and/or,” unless context clearly indicates otherwise.

As used herein, “overexpress” means to express or cause to be expresseda nucleic acid, polypeptide, or hydrocarbon in a cell at a greaterconcentration than is normally expressed in a corresponding wild-typecell. For example, a polypeptide can be “overexpressed” in a recombinanthost cell when the polypeptide is present in a greater concentration inthe recombinant host cell compared to its concentration in anon-recombinant host cell of the same species.

As used herein, “partition coefficient” or “P,” is defined as theequilibrium concentration of a compound in an organic phase divided bythe concentration at equilibrium in an aqueous phase (e.g., fermentationbroth). In one embodiment of a bi-phasic system described herein, theorganic phase is formed by the olefin during the production process.However, in some examples, an organic phase can be provided, such as byproviding a layer of octane, to facilitate product separation. Whendescribing a two phase system, the partition characteristics of acompound can be described as log P. For example, a compound with a log Pof 1 would partition 10:1 to the organic phase. A compound with a log Pof −1 would partition 1:10 to the organic phase. By choosing anappropriate fermentation broth and organic phase, an olefin with a highlog P value can separate into the organic phase even at very lowconcentrations in the fermentation vessel.

As used herein, the term “purify,” “purified,” or “purification” meansthe removal or isolation of a molecule from its environment by, forexample, isolation or separation. “Substantially purified” molecules areat least about 60% free, preferably at least about 75% free, and morepreferably at least about 90% free from other components with which theyare associated. As used herein, these terms also refer to the removal ofcontaminants from a sample. For example, the removal of contaminants canresult in an increase in the percentage of olefins in a sample. Forexample, when olefins are produced in a host cell, the olefins can bepurified by the removal of host cell proteins. After purification, thepercentage of olefins in the sample is increased.

The terms “purify,” “purified,” and “purification” do not requireabsolute purity. They are relative terms. Thus, for example, whenolefins are produced in host cells, a purified olefin is one that issubstantially separated from other cellular components (e.g., nucleicacids, polypeptides, lipids, carbohydrates, or other hydrocarbons). Inanother example, a purified olefin preparation is one in which theolefin is substantially free from contaminants, such as those that mightbe present following fermentation. In some embodiments, an olefin ispurified when at least about 50% by weight of a sample is composed ofthe olefin. In other embodiments, an olefin is purified when at leastabout 60%, 70%, 80%, 85%, 90%, 92%, 95%, 98%, or 99% or more by weightof a sample is composed of the olefin.

As used herein, the term “recombinant polypeptide” refers to apolypeptide that is produced by recombinant DNA techniques, whereingenerally DNA encoding the expressed protein or RNA is inserted into asuitable expression vector and that is in turn used to transform a hostcell to produce the polypeptide or RNA.

As used herein, the term “substantially identical” (or “substantiallyhomologous”) is used to refer to a first amino acid or nucleotidesequence that contains a sufficient number of identical or equivalent(e.g., with a similar side chain, e.g., conserved amino acidsubstitutions) amino acid residues or nucleotides to a second amino acidor nucleotide sequence such that the first and second amino acid ornucleotide sequences have similar activities.

As used herein, the term “transfection” means the introduction of anucleic acid (e.g., via an expression vector) into a recipient cell bynucleic acid-mediated gene transfer.

As used herein, “transformation” refers to a process in which a cell'sgenotype is changed as a result of the cellular uptake of exogenous DNAor RNA. This may result in the transformed cell expressing a recombinantform of an RNA or polypeptide. In the case of antisense expression fromthe transferred gene, the expression of a naturally-occurring form ofthe polypeptide is disrupted.

As used herein, a “transport protein” is a polypeptide that facilitatesthe movement of one or more compounds in and/or out of a cellularorganelle and/or a cell.

As used herein, a “variant” of polypeptide X refers to a polypeptidehaving the amino acid sequence of peptide X in which one or more aminoacid residues is altered. The variant may have conservative changes ornonconservative changes. Guidance in determining which amino acidresidues may be substituted, inserted, or deleted without affectingbiological activity may be found using computer programs well known inthe art, for example, LASERGENE software (DNASTAR).

The term “variant,” when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to that of agene or the coding sequence thereof. This definition may also include,for example, “allelic,” “splice,” “species,” or “polymorphic” variants.A splice variant may have significant identity to a referencepolynucleotide, but will generally have a greater or lesser number ofpolynucleotides due to alternative splicing of exons during mRNAprocessing. The corresponding polypeptide may possess additionalfunctional domains or an absence of domains. Species variants arepolynucleotide sequences that vary from one species to another. Theresulting polypeptides generally will have significant amino acididentity relative to each other. A polymorphic variant is a variation inthe polynucleotide sequence of a particular gene between individuals ofa given species.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of useful vector is an episome (i.e., a nucleic acidcapable of extra-chromosomal replication). Useful vectors are thosecapable of autonomous replication and/or expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids,” whichrefer generally to circular double stranded DNA loops that, in theirvector form, are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably, as theplasmid is the most commonly used form of vector. However, also includedare such other forms of expression vectors that serve equivalentfunctions and that become known in the art subsequently hereto.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A, 1B, 1C and 1D are GC/MS traces of olefins produced byJeotgalicoccus sp. ATCC 8456 cells, Jeotgalicoccus halotolerans DSMZ17274 cells, Jeotgalicoccus pinnipedalis DSMZ 17030 cells, andJeotgalicoccus psychrophilus DSMZ 19085 cells, respectively.

FIGS. 2A and 2B are mass spectrometry fragmentation patterns of twoα-olefins produced by Jeotgalicoccus ATCC 8456 cells. Compound A wasidentified as 1-nonadecene and compound B as 18-methyl-1-nonadecene.

FIG. 3 is a schematic of a phylogenetic analysis of 16s rRNA ofJeotgalicoccus ATCC 8456.

FIGS. 4A and 4B are GC/MS traces of α-olefins produced by Jeotgalicoccussp. ATCC 8456 cells upon feeding with eisosanoic acid (FIG. 4A) orstearic acid (FIG. 4B).

FIG. 5 is a GC/MS trace of α-olefins produced by cell free lysates ofJeotgalicoccus sp. ATCC 8456 cells.

FIG. 6 is a digital representation of an SDS-PAGE gel of final purifiedα-olefins-producing protein fraction from Jeotgalicoccus sp. ATCC 8456cells.

FIGS. 7A, 7B, and 7D-7N are orf880 and orf880 homolog nucleotide andamino acid sequences.

FIG. 7C is the partial 16s rRNA sequence of Jeotgalicoccus sp. ATCC8456.

FIG. 8 is a GC/MS trace of α-olefins produced by E. coli upon expressionof Jeotgalicoccus sp. 8456_orf880 and feeding of stearic acid.

FIG. 9 is a schematic of a bootstrap phylogenetic analysis of8456_orf880 homologs using ClustalW.

FIG. 10 is a table identifying various genes that can be expressed,overexpressed or attenuated to increase production of particular fattyacid substrates.

FIG. 11 is a representation of a gel of PCR products from MG1655 wildtype cells, ΔfadD::Cm cells, and ΔfadD cells.

FIG. 12A is a GC/MS trace of α-olefins produced by MG1655 ΔfadD cellstransformed with empty vector. FIG. 12B is a GC/MS trace of α-olefinsproduced by MG1655 ΔfadD cells transformed with Jeotgalicoccus sp.8456_orf880.

FIGS. 13A, 13B, and 13C are GC/MS traces of α-olefins produced by E.coli C41 ΔfadE cells transformed with empty vector (FIG. 13A),Corynebacterium efficiens YS-134 orf_CE2459 without stearic acid feeding(FIG. 13B), or with Corynebacterium efficiens YS-134 orf_CE2459 withstearic acid feeding (FIG. 13C).

FIGS. 14A, 14B, 14C, and 14D are GC/MS traces of α-olefins produced byE. coli C41 ΔfadE cells transformed with empty vector (FIG. 14A), emptyvector with stearic acid feeding (FIG. 14B), Kokuria rhizopila DC2201orf_KRH21570 without stearic acid feeding (FIG. 14C), or with Kokuriarhizopila DC2201 orf_KRH21570 with stearic acid feeding (FIG. 14D).

FIGS. 15A, 15B, and 15C are GC/MS traces of α-olefins produced by B.subtilis cells transformed with Jeotgalicoccus sp. ATCC8456_orf880 (FIG.15A), C. efficiens YS-134 orf_CE2459 (FIG. 15B), or empty vector (FIG.15C).

FIG. 16 is a set of GC/MS traces of α-olefins produced in vitro byCorynebacterium efficiens ORF_CE2459.

FIGS. 17A, 17B, and 17C are GC/MS traces of α-olefins produced in vitroby purified Kokuria rhizophila orf_KRH21570 with hexadecanoic acid assubstrate (FIG. 17A), by purified Kokuria rhizophila orf_KRH21579 withno substrate (FIG. 17B), and with no enzyme (FIG. 17C).

FIGS. 18A, 18B, and 18C are GC/MS traces of α-olefins produced in vitroby purified Methylobacterium populi orf_Mpop1292 with hexadecanoic acidas substrate (FIG. 18A), by purified Methylobacterium populiorf_Mpop1292 with no substrate (FIG. 18B), and with no enzyme (FIG.18C).

FIG. 19 is a set of GC/MS traces of α-olefins produced by E. coli cellstransformed with Bacillus subtilis P450_(Bsβ) or empty vector.

FIG. 20 is a graph demonstrating the specific activity of 1-pentadeceneand α(2)- and β(3)-hydroxy hexadecanoic acid production for ORF880,CYP152A1, and CYP152A1 Q85H using hexadecanoic acid as a substrate.

FIG. 21 is a graph demonstrating the ratio of decarboxylation tohydroxylation specific activities for ORF880, CYP152A1, and CYP152A1Q85H.

FIG. 22 is a table identifying various P450 peroxygenases that can beexpressed or overexpressed to produce α-olefins.

DETAILED DESCRIPTION

The invention provides compositions and methods of producing olefinsfrom fatty acid substrates, in particular, terminal olefins. Sucholefins are useful as a biofuel (e.g., substitutes for gasoline, diesel,jet fuel, etc.), specialty chemicals (e.g., lubricants, fuel additive,etc.), or feedstock for further chemical conversion (e.g., fuels,polymers, plastics, textiles, solvents, adhesives, etc.). The inventionis based on the discovery of a novel gene, orf880, which encodes anolefin-producing enzyme. The polynucleotide sequence of orf880 andhomologs of orf880 are presented as SEQ ID NO:1, 4, 6, 8, 9, 11, or 13in FIG. 7, and the corresponding amino acid sequences are presented asSEQ ID NO:2, 5, 7, 10, 12, or 14 in FIG. 7. Using the methods describedherein, olefins can be prepared using orf880, orf880 homologs, orvariants thereof utilizing host cells or cell-free methods.

ORF880 Polynucleotide Variants

The methods and compositions described herein include the orf880polynucleotide sequence (SEQ ID NO:1) as well as orf880 homologsdepicted in FIG. 7 (SEQ ID NO: 4, 6, 8, 9, 11, or 13) and polynucleotidevariants thereof. The variants can be naturally occurring or created invitro. In particular, such variants can be created using geneticengineering techniques, such as site directed mutagenesis, randomchemical mutagenesis, Exonuclease III deletion procedures, and standardcloning techniques. Alternatively, such variants, fragments, analogs, orderivatives can be created using chemical synthesis or modificationprocedures.

Methods of making variants are well known in the art. These includeprocedures in which nucleic acid sequences obtained from naturalisolates are modified to generate nucleic acids that encode polypeptideshaving characteristics that enhance their value in industrial orlaboratory applications. In such procedures, a large number of variantsequences having one or more nucleotide differences with respect to thesequence obtained from the natural isolate are generated andcharacterized. Typically, these nucleotide differences result in aminoacid changes with respect to the polypeptides encoded by the nucleicacids from the natural isolates.

For example, variants can be created using error prone PCR (see, e.g.,Leung et al., Technique 1:11-15, 1989; and Caldwell et al., PCR MethodsApplic. 2:28-33, 1992). In error prone PCR, PCR is performed underconditions where the copying fidelity of the DNA polymerase is low, suchthat a high rate of point mutations is obtained along the entire lengthof the PCR product. Briefly, in such procedures, nucleic acids to bemutagenized (e.g., an orf880 polynucleotide sequence), are mixed withPCR primers, reaction buffer, MgCl₂, MnCl₂, Taq polymerase, and anappropriate concentration of dNTPs for achieving a high rate of pointmutation along the entire length of the PCR product. For example, thereaction can be performed using 20 fmoles of nucleic acid to bemutagenized (e.g., an orf880 polynucleotide sequence), 30 pmole of eachPCR primer, a reaction buffer comprising 50 mM KCl, 10 mM Tris HCl (pH8.3), and 0.01% gelatin, 7 mM MgCl₂, 0.5 mM MnCl₂, 5 units of Taqpolymerase, 0.2 mM dGTP, 0.2 mM dATP, 1 mM dCTP, and 1 mM dTTP. PCR canbe performed for 30 cycles of 94° C. for 1 min, 45° C. for 1 min, and72° C. for 1 min. However, it will be appreciated that these parameterscan be varied as appropriate. The mutagenized nucleic acids are thencloned into an appropriate vector and the activities of the polypeptidesencoded by the mutagenized nucleic acids are evaluated.

Variants can also be created using oligonucleotide directed mutagenesisto generate site-specific mutations in any cloned DNA of interest.Oligonucleotide mutagenesis is described in, for example, Reidhaar-Olsonet al., Science 241:53-57, 1988. Briefly, in such procedures a pluralityof double stranded oligonucleotides bearing one or more mutations to beintroduced into the cloned DNA are synthesized and inserted into thecloned DNA to be mutagenized (e.g., an orf880 polynucleotide sequence).Clones containing the mutagenized DNA are recovered, and the activitiesof the polypeptides they encode are assessed.

Another method for generating variants is assembly PCR. Assembly PCRinvolves the assembly of a PCR product from a mixture of small DNAfragments. A large number of different PCR reactions occur in parallelin the same vial, with the products of one reaction priming the productsof another reaction. Assembly PCR is described in, for example, U.S.Pat. No. 5,965,408.

Still another method of generating variants is sexual PCR mutagenesis.In sexual PCR mutagenesis, forced homologous recombination occursbetween DNA molecules of different, but highly related, DNA sequence invitro as a result of random fragmentation of the DNA molecule based onsequence homology. This is followed by fixation of the crossover byprimer extension in a PCR reaction. Sexual PCR mutagenesis is describedin, for example, Stemmer, PNAS, USA 91:10747-10751, 1994.

Variants can also be created by in vivo mutagenesis. In someembodiments, random mutations in a nucleic acid sequence are generatedby propagating the sequence in a bacterial strain, such as an E. colistrain, which carries mutations in one or more of the DNA repairpathways. Such “mutator” strains have a higher random mutation rate thanthat of a wild-type strain. Propagating a DNA sequence (e.g., an orf880polynucleotide sequence) in one of these strains will eventuallygenerate random mutations within the DNA. Mutator strains suitable foruse for in vivo mutagenesis are described in, for example, PCTPublication No. WO 91/16427.

Variants can also be generated using cassette mutagenesis. In cassettemutagenesis, a small region of a double stranded DNA molecule isreplaced with a synthetic oligonucleotide “cassette” that differs fromthe native sequence. The oligonucleotide often contains a completelyand/or partially randomized native sequence.

Recursive ensemble mutagenesis can also be used to generate variants.Recursive ensemble mutagenesis is an algorithm for protein engineering(i.e., protein mutagenesis) developed to produce diverse populations ofphenotypically related mutants whose members differ in amino acidsequence. This method uses a feedback mechanism to control successiverounds of combinatorial cassette mutagenesis. Recursive ensemblemutagenesis is described in, for example, Arkin et al., PNAS, USA89:7811-7815, 1992.

In some embodiments, variants are created using exponential ensemblemutagenesis. Exponential ensemble mutagenesis is a process forgenerating combinatorial libraries with a high percentage of unique andfunctional mutants, wherein small groups of residues are randomized inparallel to identify, at each altered position, amino acids which leadto functional proteins. Exponential ensemble mutagenesis is describedin, for example, Delegrave et al., Biotech. Res. 11:1548-1552, 1993.Random and site-directed mutagenesis are described in, for example,Arnold, Curr. Opin. Biotech. 4:450-455, 1993.

In some embodiments, variants are created using shuffling procedureswherein portions of a plurality of nucleic acids that encode distinctpolypeptides are fused together to create chimeric nucleic acidsequences that encode chimeric polypeptides as described in, forexample, U.S. Pat. Nos. 5,965,408 and 5,939,250.

Polynucleotide variants also include nucleic acid analogs. Nucleic acidanalogs can be modified at the base moiety, sugar moiety, or phosphatebackbone to improve, for example, stability, hybridization, orsolubility of the nucleic acid. Modifications at the base moiety includedeoxyuridine for deoxythymidine and 5-methyl-2′-deoxycytidine or5-bromo-2′-doxycytidine for deoxycytidine. Modifications of the sugarmoiety include modification of the 2′ hydroxyl of the ribose sugar toform 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphatebackbone can be modified to produce morpholino nucleic acids, in whicheach base moiety is linked to a six-membered, morpholino ring, orpeptide nucleic acids, in which the deoxyphosphate backbone is replacedby a pseudopeptide backbone and the four bases are retained. (See, e.g.,Summerton et al., Antisense Nucleic Acid Drug Dev. (1997) 7:187-195; andHyrup et al., Bioorgan. Med. Chem. (1996) 4:5-23.) In addition, thedeoxyphosphate backbone can be replaced with, for example, aphosphorothioate or phosphorodithioate backbone, a phosphoroamidite, oran alkyl phosphotriester backbone.

ORF880 Polypeptide Variants

The methods and compositions described herein also include the ORF880amino acid sequence (SEQ ID NO:2) depicted in FIG. 7, homologs of ORF880(SEQ ID NO:5, 7, 10, 12, or 14) depicted in FIG. 7, and variantsthereof. ORF880 polypeptide variants can be variants in which one ormore amino acid residues are substituted with a conserved ornon-conserved amino acid residue (preferably a conserved amino acidresidue). Such substituted amino acid residue may or may not be oneencoded by the genetic code.

Conservative substitutions are those that substitute a given amino acidin a polypeptide by another amino acid of similar characteristics.Typical conservative substitutions are the following replacements:replacement of an aliphatic amino acid, such as alanine, valine,leucine, and isoleucine, with another aliphatic amino acid; replacementof a serine with a threonine or vice versa; replacement of an acidicresidue, such as aspartic acid and glutamic acid, with another acidicresidue; replacement of a residue bearing an amide group, such asasparagine and glutamine, with another residue bearing an amide group;exchange of a basic residue, such as lysine and arginine, with anotherbasic residue; and replacement of an aromatic residue, such asphenylalanine and tyrosine, with another aromatic residue.

Other polypeptide variants are those in which one or more amino acidresidues include a substituent group. Still other polypeptide variantsare those in which the polypeptide is associated with another compound,such as a compound to increase the half-life of the polypeptide (e.g.,polyethylene glycol).

Additional polypeptide variants are those in which additional aminoacids are fused to the polypeptide, such as a leader sequence, asecretory sequence, a proprotein sequence, or a sequence whichfacilitates purification, enrichment, or stabilization of thepolypeptide.

In some instances, the polypeptide variants retain the same biologicalfunction as ORF880 (e.g., retain decarboxylase activity) and have aminoacid sequences substantially identical thereto.

In other instances, the polypeptide variants have at least about 50%, atleast about 55%, at least about 60%, at least about 65%, at least about70%, at least about 75%, at least about 80%, at least about 85%, atleast about 90%, at least about 95%, or more than about 95% homology tothe amino acid sequence depicted in FIG. 7 (SEQ ID NO:2, 5, 7, 10, 12,or 14). In another embodiment, the polypeptide variants include afragment comprising at least about 5, 10, 15, 20, 25, 30, 35, 40, 50,75, 100, or 150 consecutive amino acids thereof.

The polypeptide variants or fragments thereof can be obtained byisolating nucleic acids encoding them using techniques described hereinor by expressing synthetic nucleic acids encoding them. Alternatively,polypeptide variants or fragments thereof can be obtained throughbiochemical enrichment or purification procedures. The sequence ofpolypeptide variants or fragments can be determined by proteolyticdigestion, gel electrophoresis, and/or microsequencing. The sequence ofthe polypeptide variants or fragments can then be compared to the aminoacid sequence depicted in FIG. 7 (SEQ ID NO:2, 5, 7, 10, 12, or 14)using any of the programs described herein.

The polypeptide variants and fragments thereof can be assayed forolefin-producing activity using routine methods. For example, thepolypeptide variants or fragment can be contacted with a substrate(e.g., a fatty acid substrate) under conditions that allow thepolypeptide variant to function. A decrease in the level of thesubstrate or an increase in the level of an olefin can be measured todetermine olefin-producing activity.

ORF880 Motifs

By using bioinformatics, amino acid motifs can be designed byidentifying conserved regions of the fatty acid decarboxylase (e.g.,Jeotgalicoccus sp. 8456 orf880). These amino acid motifs can be designedby methods well known in the art, such as bioinformatics, phylogeneticstudy, and/or protein alignments followed by visual inspection of theprotein sequences. These amino acid motifs can then be used to identifyproteins that have similar biological functions as the fatty aciddecarboxylase. Several programs well known in the art can use the aminoacid motifs to identify proteins that belong to the family of functionalproteins.

Based on the experimental data, it was predicted that organisms thatcontain polypeptide sequences containing these amino acid motifs may befunctional fatty acid decarboxylases

-   -   (1) F-X-X-E-[VI]-[RK]-R-X-Y-P-F-{F}-P-X-[LIV];    -   (2) P-X(6)-[LI]-X(4)-{G}-[VI]-[HQM]-X-[MLV]-D-G-X(2)-H-X(2)-R-K;    -   (3)        [AC]-[AG]-[IV]-[DE]-[IL]-X-N-X(2)-R-P-X-[VI]-A-X(3)-[FY]-X(2)-F-X(3)-A-[LMV]-X-[DE];    -   (4)        {RK}-X(6)-[LMV]-X(4)-[AC]-[AG]-[IV]-[DE]-[IL]-X-N-X(2)-R-P-X-[VI]-A-X(3)-[FY]-X(2)-F-X(3)-A-[LMV]-X-[DE].        In the sequences described herein, [AB] is A or B (e.g., [VI] is        valine or isoleucine), {A} is any amino acid except A (e.g., {F}        stands for any amino acid except Phe), X represents any amino        acid (e.g., any naturally occurring amino acid), and X(n) is X        repeated n times (e.g., X(4) is X-X-X-X).

The invention is directed to an isolated nucleic acid encoding apolypeptide comprising a fatty acid decarboxylase amino acid motifsequence. For example, the isolated nucleic acid can encode apolypeptide comprising an amino acid sequence selected from the motifsdescribed herein.

Preferably, the isolated nucleic acid encodes a polypeptide of no morethan about 700, 650, 600, 550, 500, or 450 amino acid residuescomprising a fatty acid decarboxylase amino acid motif sequence.

Anti-Orf880 Antibodies

The Orf880 polypeptides described herein can also be used to produceantibodies directed against Orf880 polypeptides. Such antibodies can beused, for example, to detect the expression of an Orf880 polypeptideusing methods known in the art. The antibody can be, e.g., a polyclonalantibody; a monoclonal antibody or antigen binding fragment thereof; amodified antibody such as a chimeric antibody, reshaped antibody,humanized antibody, or fragment thereof (e.g., Fab′, Fab, F(ab′)₂); or abiosynthetic antibody, e.g., a single chain antibody, single domainantibody (DAB), Fv, single chain Fv (scFv), or the like.

Methods of making and using polyclonal and monoclonal antibodies aredescribed, e.g., in Harlow et al., Using Antibodies: A LaboratoryManual: Portable Protocol I. Cold Spring Harbor Laboratory (Dec. 1,1998). Methods for making modified antibodies and antibody fragments(e.g., chimeric antibodies, reshaped antibodies, humanized antibodies,or fragments thereof, e.g., Fab′, Fab, F(ab′)₂ fragments); orbiosynthetic antibodies (e.g., single chain antibodies, single domainantibodies (DABs), Fv, single chain Fv (scFv), and the like), are knownin the art and can be found, e.g., in Zola, Monoclonal Antibodies:Preparation and Use of Monoclonal Antibodies and Engineered AntibodyDerivatives, Springer Verlag (Dec. 15, 2000; 1st edition).

Fatty Acid Substrates

The compositions and methods described herein can be used to produceolefins (e.g., terminal olefins) from fatty acid substrates. While notwishing to be bound by theory, it is believed that the polypeptidesdescribed herein produce olefins from fatty acid substrates via adecarboxylation mechanism. Thus, olefins having particular branchingpatterns, levels of saturation, and carbon chain length can be producedfrom fatty acid substrates having those particular characteristics.Accordingly, each step within a fatty acid biosynthetic pathway can bemodified to produce or overproduce a fatty acid substrate of interest.For example, known genes involved in the fatty acid biosynthetic pathwaycan be expressed, overexpressed, or attenuated in host cells to producea desired fatty acid substrate (see, e.g., PCT/US08/058,788). Exemplarygenes are provided in FIG. 10. It is recognized, however, that thecarbon chain of an olefin produced using the methods described hereinwill have one less carbon than the fatty acid substrate from which itwas produced.

Synthesis of Fatty Acid Substrates

Fatty acid synthase (FAS) is a group of polypeptides that catalyze theinitiation and elongation of acyl chains (Marrakchi et al., BiochemicalSociety, 30:1050-1055, 2002). The acyl carrier protein (ACP) along withthe enzymes in the FAS pathway control the length, degree of saturation,and branching of the fatty acids produced. The fatty acid biosyntheticpathway involves the precursors acetyl-CoA and malonyl-CoA. The steps inthis pathway are catalyzed by enzymes of the fatty acid biosynthesis(fab) and acetyl-CoA carboxylase (acc) gene families (see, e.g., Heathet al., Prog. Lipid Res. 40(6):467-97 (2001)).

Host cells can be engineered to express fatty acid substrates byrecombinantly expressing or overexpressing acetyl-CoA and/or malonyl-CoAsynthase genes. For example, to increase acetyl-CoA production, one ormore of the following genes can be expressed in a host cell: pdh, panK,aceEF (encoding the E1p dehydrogenase component and the E2pdihydrolipoamide acyltransferase component of the pyruvate and2-oxoglutarate dehydrogenase complexes), fabH, fabD, fabG, acpP, andfabF. Exemplary GenBank accession numbers for these genes are: pdh(BAB34380, AAC73227, AAC73226), panK (also known as coaA, AAC76952),aceEF (AAC73227, AAC73226), fabH (AAC74175), fabD (AAC74176), fabG(AAC74177), acpP (AAC74178), fabF (AAC74179). Additionally, theexpression levels of fadE, gpsA, ldhA, pflb, adhE, pta, poxB, ackA,and/or ackB can be attenuated or knocked-out in an engineered host cellby transformation with conditionally replicative or non-replicativeplasmids containing null or deletion mutations of the correspondinggenes or by substituting promoter or enhancer sequences. ExemplaryGenBank accession numbers for these genes are: fadE (AAC73325), gspA(AAC76632), ldhA (AAC74462), pflb (AAC73989), adhE (AAC74323), pta(AAC75357), poxB (AAC73958), ackA (AAC75356), and ackB (BAB81430). Theresulting host cells will have increased acetyl-CoA production levelswhen grown in an appropriate environment.

Malonyl-CoA overexpression can be effected by introducing accABCD (e.g.,accession number AAC73296, EC 6.4.1.2) into a host cell. Fatty acids canbe further overexpressed in host cells by introducing into the host cella DNA sequence encoding a lipase (e.g., accession numbers CAA89087,CAA98876).

In addition, inhibiting PlsB can lead to an increase in the levels oflong chain acyl-ACP, which will inhibit early steps in the pathway(e.g., accABCD, fabH, and fabI). The plsB (e.g., accession numberAAC77011) D311E mutation can be used to increase the amount of availableacyl-CoA.

In addition, a host cell can be engineered to overexpress a sfa gene(suppressor of fabA, e.g., accession number AAN79592) to increaseproduction of monounsaturated fatty acids (Rock et al., J. Bacteriology178:5382-5387, 1996).

In some instances, host cells can be engineered to express, overexpress,or attenuate expression of a thioesterase to increase fatty acidsubstrate production. The chain length of a fatty acid substrate iscontrolled by thioesterase. In some instances, a tes or fat gene can beoverexpressed. In other instances, C₁₀ fatty acids can be produced byattenuating thioesterase C₁₈ (e.g., accession numbers AAC73596 andP0ADA1), which uses C_(18:1)-ACP, and expressing thioesterase C₁₀ (e.g.,accession number Q39513), which uses C₁₋₁₀-ACP. This results in arelatively homogeneous population of fatty acids that have a carbonchain length of 10. In yet other instances, C₁₄ fatty acids can beproduced by attenuating endogenous thioesterases that produce non-C₁₄fatty acids and expressing the thioesterases, that use C₁₄-ACP (forexample, accession number Q39473). In some situations, C₁₂ fatty acidscan be produced by expressing thioesterases that use C₁₂-ACP (forexample, accession number Q41635) and attenuating thioesterases thatproduce non-C₁₂ fatty acids. Acetyl-CoA, malonyl-CoA, and fatty acidoverproduction can be verified using methods known in the art, forexample, by using radioactive precursors, HPLC, and GC-MS subsequent tocell lysis. Non-limiting examples of thioesterases that can be used inthe methods described herein are listed in Table 1.

TABLE 1 Thioesterases Preferential Accession Number Source Organism Geneproduct produced AAC73596 E. coli tesA without C_(18:1) leader sequenceAAC73555 E. coli tesB Q41635, AAA34215 Umbellularia california fatBC_(12:0) Q39513; AAC49269 Cuphea hookeriana fatB2 C_(8:0)-C_(10:0)AAC49269; AAC72881 Cuphea hookeriana fatB3 C_(14:0)-C_(16:0) Q39473,AAC49151 Cinnamonum camphorum fatB C_(14:0) CAA85388 Arabidopsisthaliana fatB [M141T]* C_(16:1) NP 189147; NP 193041 Arabidopsisthaliana fatA C_(18:1) CAC39106 Bradyrhiizobium japonicum fatA C_(18:1)AAC72883 Cuphea hookeriana fatA C_(18:1) AAL79361 Helianthus annus fatA1*Mayer et al., BMC Plant Biology 7: 1-11, 2007Formation of Branched Olefins

Olefins can be produced that contain branch points by using branchedfatty acids as substrates. For example, although E. coli naturallyproduces straight chain fatty acids (sFAs), E. coli can be engineered toproduce branched chain fatty acids (brFAs) by introducing and expressingor overexpressing genes that provide branched precursors in the E. coli(e.g., bkd, ilv, icm, and fab gene families). Additionally, a host cellcan be engineered to express or overexpress genes encoding proteins forthe elongation of brFAs (e.g., ACP, FabF, etc.) and/or to delete orattenuate the corresponding host cell genes that normally lead to sFAs.

The first step in forming brFAs is the production of the correspondingα-keto acids by a branched-chain amino acid aminotransferase. Host cellsmay endogenously include genes encoding such enzymes or such genes canbe recombinantly introduced. E. coli, for example, endogenouslyexpresses such an enzyme, IlvE (EC 2.6.1.42; GenBank accessionYP_(—)026247). In some host cells, a heterologous branched-chain aminoacid aminotransferase may not be expressed. However, E. coli ENE or anyother branched-chain amino acid aminotransferase (e.g., IlvE fromLactococcus lactis (GenBank accession AAF34406), IlvE from Pseudomonasputida (GenBank accession NP_(—)745648), or IlvE from Streptomycescoelicolor (GenBank accession NP_(—)629657)), if not endogenous, can beintroduced.

The second step is the oxidative decarboxylation of the α-ketoacids tothe corresponding branched-chain acyl-CoA. This reaction can becatalyzed by a branched-chain α-keto acid dehydrogenase complex (bkd; EC1.2.4.4.) (Denoya et al., J. Bacteriol. 177:3504, 1995), which consistsof E1α/β (decarboxylase), E2 (dihydrolipoyl transacylase), and E3(dihydrolipoyl dehydrogenase) subunits. These branched-chain α-keto aciddehydrogenase complexes are similar to pyruvate and α-ketoglutaratedehydrogenase complexes. Any microorganism that possesses brFAs and/orgrows on branched-chain amino acids can be used as a source to isolatebkd genes for expression in host cells, for example, E. coli.Furthermore, E. coli has the E3 component as part of its pyruvatedehydrogenase complex (lpd, EC 1.8.1.4, GenBank accession NP_(—)414658).Thus, it can be sufficient to express only the E1 α/β and E2 bkd genes.Table 2 lists non-limiting examples of bkd genes from severalmicroorganisms that can be recombinantly introduced and expressed in ahost cell to provide branched-chain acyl-CoA precursors.

TABLE 2 Bkd genes from selected microorganisms GenBank Organism GeneAccession # Streptomyces coelicolor bkdA1 (E1α) NP_628006 bkdB1 (E1β)NP_628005 bkdC1 (E2) NP_638004 Streptomyces coelicolor bkdA2 (E1α)NP_733618 bkdB2 (E1β) NP_628019 bkdC2 (E2) NP_628018 Streptomycesavermitilis bkdA (E1a) BAC72074 bkdB (E1b) BAC72075 bkdC (E2) BAC72076Streptomyces avermitilis bkdF (E1α) BAC72088 bkdG (E1β) BAC72089 bkdH(E2) BAC72090 Bacillus subtilis bkdAA (E1α) NP_390288 bkdAB (E1β)NP_390288 bkdB (E2) NP_390288 Pseudomonas putida bkdA1 (E1α) AAA65614bkdA2 (E1β) AAA65615 bkdC (E2) AAA65617

In another example, isobutyryl-CoA can be made in a host cell, forexample in E. coli, through the coexpression of a crotonyl-CoA reductase(Ccr, EC 1.6.5.5, 1.1.1.1) and isobutyryl-CoA mutase (large subunitIcmA, EC 5.4.99.2; small subunit IcmB, EC 5.4.99.2) (Han and Reynolds,J. Bacteriol. 179:5157, 1997). Crotonyl-CoA is an intermediate in fattyacid biosynthesis in E. coli and other microorganisms. Non-limitingexamples of ccr and icm genes from selected microorganisms are listed inTable 3.

TABLE 3 Ccr and icm genes from selected microorganisms GenBank OrganismGene Accession # Streptomyces coelicolor Ccr NP_630556 icmA NP_629554icmB NP_630904 Streptomyces cinnamonensis ccr AAD53915 icmA AAC08713icmB AJ246005

In addition to expression of the bkd genes, the initiation of brFAbiosynthesis utilizes β-ketoacyl-acyl-carrier-protein synthase III(FabH, EC 2.3.1.41) with specificity for branched chain acyl-CoAs (Li etal., J. Bacteriol. 187:3795-3799, 2005). Non-limiting examples of suchFabH enzymes are listed in Table 4. fabH genes that are involved infatty acid biosynthesis of any brFA-containing microorganism can beexpressed in a host cell. The Bkd and FabH enzymes from host cells thatdo not naturally make brFA may not support brFA production. Therefore,bkd and fabH can be expressed recombinantly. Vectors containing the bkdand fabH genes can be inserted into such a host cell. Similarly, theendogenous level of Bkd and FabH production may not be sufficient toproduce brFA. In this case, they can be overexpressed. Additionally,other components of the fatty acid biosynthesis pathway can be expressedor overexpressed, such as acyl carrier proteins (ACPs) andβ-ketoacyl-acyl-carrier-protein synthase II (fabF, EC 2.3.1.41)(non-limiting examples of candidates are listed in Table 4). In additionto expressing these genes, some genes in the endogenous fatty acidbiosynthesis pathway can be attenuated in the host cell (e.g., the E.coli genes fabH (GenBank accession #NP_(—)415609) and/or fabF (GenBankaccession #NP_(—)415613)).

TABLE 4 FabH, ACP and fabF genes from selected microorganisms with brFAsGenBank Organism Gene Accession # Streptomyces coelicolor fabH1NP_626634 ACP NP_626635 fabF NP_626636 Streptomyces avermitilis fabH3NP_823466 fabC3 (ACP) NP_823467 fabF NP_823468 Bacillus subtilis fabH_ANP_389015 fabH_B NP_388898 ACP NP_389474 fabF NP_389016 StenotrophomonasSmalDRAFT_0818 (FabH) ZP_01643059 maltophilia SmalDRAFT_0821 (ACP)ZP_01643063 SmalDRAFT_0822 (FabF) ZP_01643064 Legionella pneumophilaFabH YP_123672 ACP YP_123675 fabF YP_123676Formation of Cyclic Olefins

Cyclic olefins can be produced by using cyclic fatty acids assubstrates. To produce cyclic fatty acid substrates, genes that providecyclic precursors (e.g., the ans, chc, and plm gene families) can beintroduced into the host cell and expressed to allow initiation of fattyacid biosynthesis from cyclic precursors. For example, to convert a hostcell, such as E. coli, into one capable of synthesizing ω-cyclic fattyacids (cyFA), a gene that provides the cyclic precursorcyclohexylcarbonyl-CoA (CHC-CoA) (Cropp et al., Nature Biotech.18:980-983, 2000) can be introduced and expressed in the host cell.Non-limiting examples of genes that provide CHC-CoA in E. coli include:ansJ, ansK, ansL, chcA, and ansM from the ansatrienin gene cluster ofStreptomyces collinus (Chen et al., Eur. J. Biochem. 261: 98-107, 1999)or plmJ, plmK, plmL, chcA, and plmM from the phoslactomycin B genecluster of Streptomyces sp. HK803 (Palaniappan et al., J. Biol. Chem.278:35552-35557, 2003) together with the chcB gene (Patton et al.,Biochem. 39:7595-7604, 2000) from S. collinus, S. avermitilis, or S.coelicolor (see Table 5). The genes listed in Table 4 can then beexpressed to allow initiation and elongation of ω-cyclic fatty acids.Alternatively, the homologous genes can be isolated from microorganismsthat make cyFA and expressed in a host cell (e.g., E. coli).

TABLE 5 Genes for the synthesis of CHC-CoA GenBank Organism GeneAccession # Streptomyces collinus ansJK U72144* ansL chcA ansM chcBAF268489 Streptomyces sp. HK803 pmLJK AAQ84158 pmLL AAQ84159 chcAAAQ84160 pmLM AAQ84161 Streptomyces coelicolor chcB/caiD NP_629292Streptomyces avermitilis chcB/caiD NP_629292 *Only chcA is annotated inGenBank entry U72144, ansJKLM are according to Chen et al. (Eur. J.Biochem. 261: 98-107, 1999).

The genes listed in Table 4 (fabH, ACP, and fabF) allow initiation andelongation of ω-cyclic fatty acids because they have broad substratespecificity. If the coexpression of any of these genes with the geneslisted in Table 5 does not yield cyFA, then fabH, ACP, and/or fabFhomologs from microorganisms that make cyFAs (e.g., those listed inTable 6) can be isolated (e.g., by using degenerate PCR primers orheterologous DNA sequence probes) and coexpressed.

TABLE 6 Non-limiting examples of microorganisms that contain ω-cyclicfatty acids Organism Reference Curtobacterium pusillum ATCC19096Alicyclobacillus acidoterrestris ATCC49025 Alicyclobacillusacidocaldarius ATCC27009 Alicyclobacillus cycloheptanicus * Moore, J.Org. Chem. 62: pp. 2173, 1997 * Uses cycloheptylcarbonyl-CoA and notcyclohexylcarbonyl-CoA as precursor for cyFA biosynthesis.Controlling Degree of Saturation in Olefins

The degree of saturation in olefins can be controlled by regulating thedegree of saturation of olefin intermediates, for example, fatty acids.The sfa, gns, and fab families of genes can be expressed oroverexpressed to control the saturation of fatty acids. For example,host cells can be engineered to produce unsaturated fatty acids byengineering the host cells to overexpress fabB or by growing the hostcells at low temperatures (e.g., less than 37° C.). FabB has preferencefor cis-δ³decenoyl-ACP and results in unsaturated fatty acid productionin E. coli. Overexpression of fabB results in the production of asignificant percentage of unsaturated fatty acids (de Mendoza et al., J.Biol. Chem., 258:2098-101, 1983). fabB may be inserted into andexpressed in host cells not naturally having the gene. These unsaturatedfatty acids can then be used as substrates in host cells that areengineered to produce olefins.

Alternatively, a repressor of fatty acid biosynthesis, for example, fabR(GenBank accession NP_(—)418398), can be deleted, which will result inincreased unsaturated fatty acid production in E. coli (Zhang et al., J.Biol. Chem. 277:15558, 2002). Similar deletions may be made in otherhost cells. A further increase in unsaturated fatty acids can beachieved, for example, by overexpressing fabM (trans-2,cis-3-decenoyl-ACP isomerase, GenBank accession DAA05501) and controlledexpression of fabK (trans-2-enoyl-ACP reductase II, GenBank accessionNP_(—)357969) from Streptococcus pneumoniae (Marrakchi et al., J. Biol.Chem. 277: 44809, 2002), while deleting E. coli fabI (trans-2-enoyl-ACPreductase, GenBank accession NP_(—)415804). In some examples, theendogenous fabF gene can be attenuated. Thus, increasing the percentageof palmitoleate (C_(16:1)) produced.

Genetic Engineering of Host Cells to Express Olefins

Various host cells can be used to produce olefins, as described herein.A host cell can be any prokaryotic or eukaryotic cell. For example, apolypeptide described herein can be expressed in bacterial cells, suchas E. coli, insect cells, yeast or mammalian cells (such as Chinesehamster ovary cells (CHO) cells, COS cells, VERO cells, BHK cells, HeLacells, Cv1 cells, MDCK cells, 293 cells, 3T3 cells, or PC12 cells).Other exemplary host cells include cells from the members of the genusEscherichia, Bacillus, Lactobacillus, Rhodococcus, Pseudomonas,Aspergillus, Trichoderma, Neurospora, Fusarium, Humicola, Rhizomucor,Kluyveromyces, Pichia, Mucor, Myceliophtora, Penicillium, Phanerochaete,Pleurotus, Trametes, Chrysosporium, Saccharomyces, Schizosaccharomyces,Yarrowia, or Streptomyces. Yet other exemplary host cells can be aBacillus lentus cell, a Bacillus brevis cell, a Bacillusstearothermophilus cell, a Bacillus licheniformis cell, a Bacillusalkalophilus cell, a Bacillus coagulans cell, a Bacillus circulans cell,a Bacillus pumilis cell, a Bacillus thuringiensis cell, a Bacillusclausii cell, a Bacillus megaterium cell, a Bacillus subtilis cell, aBacillus amyloliquefaciens cell, a Trichoderma koningii cell, aTrichoderma viride cell, a Trichoderma reesei cell, a Trichodermalongibrachiatum cell, an Aspergillus awamori cell, an Aspergillusfumigates cell, an Aspergillus foetidus cell, an Aspergillus nidulanscell, an Aspergillus niger cell, an Aspergillus oryzae cell, a Humicolainsolens cell, a Humicola lanuginose cell, a Rhizomucor miehei cell, aMucor michei cell, a Streptomyces lividans cell, a Streptomyces murinuscell, or an Actinomycetes cell. In a preferred embodiment, the host cellis an E. coli cell. In a more preferred embodiment, the host cell isfrom E. coli strains B, C, K, or W. Other suitable host cells are knownto those skilled in the art.

Various methods well known in the art can be used to geneticallyengineer host cells to produce olefins. The methods include the use ofvectors, preferably expression vectors, containing a nucleic acidencoding an ORF880 polypeptide, polypeptide variant, or a fragmentthereof. As used herein, the term “vector” refers to a nucleic acidmolecule capable of transporting another nucleic acid to which it hasbeen linked. One type of vector is a “plasmid,” which refers to acircular double stranded DNA loop into which additional DNA segments canbe ligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell and are thereby replicatedalong with the host genome. Moreover, certain vectors, such asexpression vectors, are capable of directing the expression of genes towhich they are operatively linked. In general, expression vectors usedin recombinant DNA techniques are often in the form of plasmids.However, other forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses, and adeno-associatedviruses), can also be used.

The recombinant expression vectors described herein include a nucleicacid described herein in a form suitable for expression of the nucleicacid in a host cell. The recombinant expression vectors can include oneor more control sequences, selected on the basis of the host cell to beused for expression. The control sequence is operably linked to thenucleic acid sequence to be expressed. Such control sequences aredescribed, for example, in Goeddel, Gene Expression Technology Methodsin Enzymology 185, Academic Press, San Diego, Calif. (1990). Controlsequences include those that direct constitutive expression of anucleotide sequence in many types of host cells and those that directexpression of the nucleotide sequence only in certain host cells (e.g.,tissue-specific regulatory sequences). It will be appreciated by thoseskilled in the art that the design of the expression vector can dependon such factors as the choice of the host cell to be transformed, thelevel of expression of protein desired, etc. The expression vectorsdescribed herein can be introduced into host cells to producepolypeptides, including fusion polypeptides, encoded by the nucleicacids as described herein.

Recombinant expression vectors can be designed for expression of anORF880 polypeptide or variant in prokaryotic or eukaryotic cells (e.g.,bacterial cells, such as E. coli, insect cells (using baculovirusexpression vectors), yeast cells, or mammalian cells). Suitable hostcells are discussed further in Goeddel, Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).Alternatively, the recombinant expression vector can be transcribed andtranslated in vitro, for example, by using T7 promoter regulatorysequences and T7 polymerase.

Expression of polypeptides in prokaryotes, for example, E. coli, is mostoften carried out with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionpolypeptides. Fusion vectors add a number of amino acids to apolypeptide encoded therein, usually to the amino terminus of therecombinant polypeptide. Such fusion vectors typically serve threepurposes: (1) to increase expression of the recombinant polypeptide; (2)to increase the solubility of the recombinant polypeptide; and (3) toaid in the purification of the recombinant polypeptide by acting as aligand in affinity purification. Often, in fusion expression vectors, aproteolytic cleavage site is introduced at the junction of the fusionmoiety and the recombinant polypeptide. This enables separation of therecombinant polypeptide from the fusion moiety after purification of thefusion polypeptide. Examples of such enzymes, and their cognaterecognition sequences, include Factor Xa, thrombin, and enterokinase.Exemplary fusion expression vectors include pGEX (Pharmacia Biotech Inc;Smith et al., Gene (1988) 67:31-40), pMAL (New England Biolabs, Beverly,Mass.), and pRITS (Pharmacia, Piscataway, N.J.), which fuse glutathioneS-transferase (GST), maltose E binding protein, or protein A,respectively, to the target recombinant polypeptide.

Examples of inducible, non-fusion E. coli expression vectors includepTrc (Amann et al., Gene (1988) 69:301-315) and pET 11d (Studier et al.,Gene Expression Technology: Methods in Enzymology 185, Academic Press,San Diego, Calif. (1990) 60-89). Target gene expression from the pTrcvector relies on host RNA polymerase transcription from a hybrid trp-lacfusion promoter. Target gene expression from the pET 11d vector relieson transcription from a T7 gn10-lac fusion promoter mediated by acoexpressed viral RNA polymerase (T7 gn1). This viral polymerase issupplied by host strains BL21(DE3) or HMS174(DE3) from a resident λprophage harboring a T7 gn1 gene under the transcriptional control ofthe lacUV 5 promoter.

One strategy to maximize recombinant polypeptide expression is toexpress the polypeptide in a host cell with an impaired capacity toproteolytically cleave the recombinant polypeptide (see Gottesman, GeneExpression Technology: Methods in Enzymology 185, Academic Press, SanDiego, Calif. (1990) 119-128). Another strategy is to alter the nucleicacid sequence to be inserted into an expression vector so that theindividual codons for each amino acid are those preferentially utilizedin the host cell (Wada et al., Nucleic Acids Res. (1992) 20:2111-2118).Such alteration of nucleic acid sequences can be carried out by standardDNA synthesis techniques.

In another embodiment, the host cell is a yeast cell. In thisembodiment, the expression vector is a yeast expression vector. Examplesof vectors for expression in yeast S. cerevisiae include pYepSec1(Baldari et al., EMBO J. (1987) 6:229-234), pMFa (Kurjan et al., Cell(1982) 30:933-943), pJRY88 (Schultz et al., Gene (1987) 54:113-123),pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InvitrogenCorp, San Diego, Calif.).

Alternatively, a polypeptide described herein can be expressed in insectcells using baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include, for example, the pAc series (Smith et al., Mol. Cell.Biol. (1983) 3:2156-2165) and the pVL series (Lucklow et al., Virology(1989) 170:31-39).

In yet another embodiment, the nucleic acids described herein can beexpressed in mammalian cells using a mammalian expression vector.Examples of mammalian expression vectors include pCDM8 (Seed, Nature(1987) 329:840) and pMT2PC (Kaufman et al., EMBO J. (1987) 6:187-195).When used in mammalian cells, the expression vector's control functionscan be provided by viral regulatory elements. For example, commonly usedpromoters are derived from polyoma, Adenovirus 2, cytomegalovirus andSimian Virus 40. Other suitable expression systems for both prokaryoticand eukaryotic cells are described in chapters 16 and 17 of Sambrook etal., eds., Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold SpringHarbor Laboratory, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y., 1989.

Vectors can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” refer to a variety ofart-recognized techniques for introducing foreign nucleic acid (e.g.,DNA) into a host cell, including calcium phosphate or calcium chlorideco-precipitation, DEAE-dextran-mediated transfection, lipofection, orelectroporation. Suitable methods for transforming or transfecting hostcells can be found in, for example, Sambrook et al. (supra).

For stable transformation of bacterial cells, it is known that,depending upon the expression vector and transformation technique used,only a small fraction of cells will take-up and replicate the expressionvector. In order to identify and select these transformants, a gene thatencodes a selectable marker (e.g., resistance to antibiotics) can beintroduced into the host cells along with the gene of interest.Selectable markers include those that confer resistance to drugs, suchas ampicillin, kanamycin, chloramphenicol, or tetracycline. Nucleicacids encoding a selectable marker can be introduced into a host cell onthe same vector as that encoding a polypeptide described herein or canbe introduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) can be introducedinto the host cells along with the gene of interest. Preferredselectable markers include those which confer resistance to drugs, suchas G418, hygromycin, and methotrexate. Nucleic acids encoding aselectable marker can be introduced into a host cell on the same vectoras that encoding a polypeptide described herein or can be introduced ona separate vector. Cells stably transfected with the introduced nucleicacid can be identified by drug selection (e.g., cells that haveincorporated the selectable marker gene will survive, while the othercells die).

In certain instances, an ORF880 polypeptide, polypeptide variant, or afragment thereof, is produced in a host cell that contains a naturallyoccurring mutation that results in an increased level of fatty acids inthe host cells. In other instances, the host cell is geneticallyengineered to increase the level of fatty acids in the host cellrelative to a corresponding wild-type host cell. For example, the hostcell can be genetically engineered to express a reduced level of anacyl-CoA synthase relative to a corresponding wild-type host cell. Inone embodiment, the level of expression of one or more genes, e.g., anacyl-CoA synthase gene, is reduced by genetically engineering a “knockout” host cell.

“Gene knockout” refers to a procedure by which the gene encoding thetarget protein is modified or inactivated so to reduce or eliminate thefunction of the intact protein. Inactivation of the gene may beperformed by general methods such as mutagenesis by UV irradiation ortreatment with N-methyl-N′-nitro-N-nitrosoguanidine, site-directedmutagenesis, homologous recombination, insertion-deletion mutagenesis,or “Red-driven integration” (Datsenko et al., Proc. Natl. Acad. Sci.USA, 97:6640-45, 2000). For example, in one embodiment, a construct isintroduced into a host cell, such that it is possible to select forhomologous recombination events in the host cell. One of skill in theart can readily design a knock-out construct including both positive andnegative selection genes for efficiently selecting transfected cellsthat undergo a homologous recombination event with the construct. Thealteration in the host cell may be obtained, e.g., by replacing througha single or double crossover recombination a wild type DNA sequence by aDNA sequence containing the alteration. For convenient selection oftransformants, the alteration may, e.g., be a DNA sequence encoding anantibiotic resistance marker or a gene complementing a possibleauxotrophy of the host cell. Mutations include, but are not limited to,deletion-insertion mutations. An example of such an alteration includesa gene disruption, i.e., a perturbation of a gene such that the productthat is normally produced from this gene is not produced in a functionalform. This could be due to a complete deletion, a deletion and insertionof a selective marker, an insertion of a selective marker, a frameshiftmutation, an in-frame deletion, or a point mutation that leads topremature termination. In some instances, the entire mRNA for the geneis absent. In other situations, the amount of mRNA produced varies.

Any known acyl-CoA synthase gene can be reduced or knocked out in a hostcell. Non-limiting examples of acyl-CoA synthase genes include fadD,fadK, BH3103, yhfL, Pfl-4354, EAV15023, fadD1, fadD2, RPC_(—)4074,fadDD35, fadDD22, faa3p or the gene encoding the protein ZP_(—)01644857.Specific examples of acyl-CoA synthase genes include fadDD35 from M.tuberculosis H37Rv [NP_(—)217021], fadDD22 from M. tuberculosis H37Rv[NP_(—)217464], fadD from E. coli [NP_(—)416319], fadK from E. coli[YP_(—)416216], fadD from Acinetobacter sp. ADP1 [YP_(—)045024], fadDfrom Haemophilus influenza RdkW20 [NP_(—)438551], fadD fromRhodopseudomonas palustris Bis B18 [YP_(—)533919], BH3101 from Bacillushalodurans C-125 [NP_(—)243969], Pfl-4354 from Pseudomonas fluorescensPfo-1 [YP_(—)350082], EAV15023 from Comamonas testosterone KF-1[ZP_(—)01520072], yhfL from B. subtilis [NP_(—)388908], fadD1 from P.aeruginosa PAO1 [NP_(—)251989], fadD1 from Ralstonia solanacearum GM11000 [NP_(—)520978], fadD2 from P. aeruginosa PAO1 [NP_(—)251990], thegene encoding the protein ZP_(—)01644857 from Stenotrophomonasmaltophilia R551-3, faa3p from Saccharomyces cerevisiae [NP_(—)012257],faa1p from Saccharomyces cerevisiae [NP_(—)014962], lcfA from Bacillussubtilis [CAA99571], or those described in Shockey et al., Plant.Physiol. 129:1710-1722, 2002; Caviglia et al., J. Biol. Chem.279:1163-1169, 2004; Knoll et al., J. Biol. Chem. 269(23):16348-56,1994; Johnson et al., J. Biol. Chem. 269: 18037-18046, 1994; and Blacket al., J. Biol. Chem. 267: 25513-25520, 1992.

Transport Proteins

Transport proteins can export polypeptides and hydrocarbons (e.g.,olefins) out of a host cell. Many transport and efflux proteins serve toexcrete a wide variety of compounds and can be naturally modified to beselective for particular types of hydrocarbons.

Non-limiting examples of suitable transport proteins are ATP-BindingCassette (ABC) transport proteins, efflux proteins, and fatty acidtransporter proteins (FATP). Additional non-limiting examples ofsuitable transport proteins include the ABC transport proteins fromorganisms such as Caenorhabditis elegans, Arabidopsis thalania,Alkaligenes eutrophus, and Rhodococcus erythropolis. Exemplary ABCtransport proteins that can be used are listed in FIG. 10 (e.g., CER5,AtMRP5, AmiS2, and AtPGP1). Host cells can also be chosen for theirendogenous ability to secrete hydrocarbons. The efficiency ofhydrocarbon production and secretion into the host cell environment(e.g., culture medium, fermentation broth) can be expressed as a ratioof intracellular product to extracellular product. In some examples, theratio can be about 5:1, 4:1, 3:1, 2:1, 1:1, 1:2, 1:3, 1:4, or 1:5.

Fermentation

The production and isolation of olefins can be enhanced by employingbeneficial fermentation techniques. One method for maximizing productionwhile reducing costs is increasing the percentage of the carbon sourcethat is converted to hydrocarbon products.

During normal cellular lifecycles, carbon is used in cellular functions,such as producing lipids, saccharides, proteins, organic acids, andnucleic acids. Reducing the amount of carbon necessary forgrowth-related activities can increase the efficiency of carbon sourceconversion to product. This can be achieved by, for example, firstgrowing host cells to a desired density (for example, a density achievedat the peak of the log phase of growth). At such a point, replicationcheckpoint genes can be harnessed to stop the growth of cells.Specifically, quorum sensing mechanisms (reviewed in Camilli et al.,Science 311:1113, 2006; Venturi FEMS Microbio. Rev. 30:274-291, 2006;and Reading et al., FEMS Microbiol. Lett. 254:1-11, 2006) can be used toactivate checkpoint genes, such as p53, p21, or other checkpoint genes.

Genes that can be activated to stop cell replication and growth in E.coli include umuDC genes. The overexpression of umuDC genes stops theprogression from stationary phase to exponential growth (Murli et al.,J. of Bact. 182:1127, 2000). UmuC is a DNA polymerase that can carry outtranslesion synthesis over non-coding lesions—the mechanistic basis ofmost UV and chemical mutagenesis. The umuDC gene products are involvedin the process of translesion synthesis and also serve as a DNA sequencedamage checkpoint. The umuDC gene products include UmuC, UmuD, umuD′,UmuD′₂C, UmuD′₂, and UmuD₂. Simultaneously, product-producing genes canbe activated, thus minimizing the need for replication and maintenancepathways to be used while an olefin is being made.

The percentage of input carbons converted to olefins can be a costdriver. The more efficient the process is (i.e., the higher thepercentage of input carbons converted to olefins), the less expensivethe process will be. For oxygen-containing carbon sources (e.g., glucoseand other carbohydrate based sources), the oxygen must be released inthe form of carbon dioxide. For every 2 oxygen atoms released, a carbonatom is also released leading to a maximal theoretical metabolicefficiency of approximately 34% (w/w) (for fatty acid derived products).This figure, however, changes for other hydrocarbon products and carbonsources. Typical efficiencies in the literature are approximately lessthan 5%. Host cells engineered to produce olefins can have greater thanabout 1, 3, 5, 10, 15, 20, 25, and 30% efficiency. In one example, hostcells can exhibit an efficiency of about 10% to about 25%. In otherexamples, such host cells can exhibit an efficiency of about 25% toabout 30%. In other examples, host cells can exhibit greater than 30%efficiency.

The host cell can be additionally engineered to express recombinantcellulosomes, such as those described in PCT application numberPCT/US2007/003736. These cellulosomes can allow the host cell to usecellulosic material as a carbon source. For example, the host cell canbe additionally engineered to express invertases (EC 3.2.1.26) so thatsucrose can be used as a carbon source. Similarly, the host cell can beengineered using the teachings described in U.S. Pat. Nos. 5,000,000;5,028,539; 5,424,202; 5,482,846; and 5,602,030; so that the host cellcan assimilate carbon efficiently and use cellulosic materials as carbonsources.

In one example, the fermentation chamber can enclose a fermentation thatis undergoing a continuous reduction. In this instance, a stablereductive environment can be created. The electron balance can bemaintained by the release of carbon dioxide (in gaseous form). Effortsto augment the NAD/H and NADP/H balance can also facilitate instabilizing the electron balance. The availability of intracellularNADPH can also be enhanced by engineering the host cell to express anNADH:NADPH transhydrogenase. The expression of one or more NADH:NADPHtranshydrogenases converts the NADH produced in glycolysis to NADPH,which can enhance the production of olefins.

For small scale production, the engineered host cells can be grown inbatches of, for example, around 100 mL, 500 mL, 1 L, 2 L, 5 L, or 10 L;fermented; and induced to express desired olefins based on the specificgenes encoded in the appropriate plasmids. For example, E. coliBL21(DE3) cells harboring pBAD24 (with ampicillin resistance and theolefin synthesis pathway) as well as pUMVC1 (with kanamycin resistanceand the acetyl CoA/malonyl CoA overexpression system) can be incubatedovernight in 2 L flasks at 37° C. shaken at >200 rpm in 500 mL LB mediumsupplemented with 75 μg/mL ampicillin and 50 μg/mL kanamycin untilcultures reach an OD₆₀₀ of >0.8. Upon achieving an OD₆₀₀ of >0.8, thecells can be supplemented with 25 mM sodium proprionate (pH 8.0) toactivate the engineered gene systems for production and to stop cellularproliferation by activating UmuC and UmuD proteins. Induction can beperformed for 6 h at 30° C. After incubation, the media can be examinedfor olefins using GC-MS.

For large scale production, the engineered host cells can be grown inbatches of 10 L, 100 L, 1000 L or larger; fermented; and induced toexpress desired olefins based on the specific genes encoded in theappropriate plasmids. For example, E. coli BL21(DE3) cells harboringpBAD24 (with ampicillin resistance and the olefin synthesis pathway) aswell as pUMVC1 (with kanamycin resistance and the acetyl-CoA/malonyl-CoAoverexpression system) can be incubated from a 500 mL seed culture for10 L fermentations (5 L for 100 L fermentations, etc.) in LB media(glycerol free) with 50 μg/mL kanamycin and 75 μg/mL ampicillin at 37°C., and shaken at >200 rpm until cultures reach an OD600 of >0.8(typically 16 h). Media can be continuously supplemented to maintain 25mM sodium proprionate (pH 8.0) to activate the engineered gene systemsfor production and to stop cellular proliferation by activating umuC andumuD proteins. Media can be continuously supplemented with glucose tomaintain a concentration 25 g/100 mL.

After the first hour of induction, aliquots of no more than 10% of thetotal cell volume can be removed each hour and allowed to sit withoutagitation to allow the olefins to rise to the surface and undergo aspontaneous phase separation. The olefin component can then becollected, and the aqueous phase returned to the reaction chamber. Thereaction chamber can be operated continuously. When the OD₆₀₀ dropsbelow 0.6, the cells can be replaced with a new batch grown from a seedculture.

Producing Olefins Using Cell-Free Methods

In some methods described herein, an olefin can be produced using apurified polypeptide described herein and a fatty acid substrate. Forexample, a host cell can be engineered to express a polypeptide (e.g.,an ORF880 polypeptide or variant) as described herein. The host cell canbe cultured under conditions suitable to allow expression of thepolypeptide. Cell free extracts can then be generated using knownmethods. For example, the host cells can be lysed using detergents or bysonication. The expressed polypeptides can be purified using knownmethods. After obtaining the cell free extracts, fatty acid substratesdescribed herein can be added to the cell free extracts and maintainedunder conditions to allow conversion of the fatty acid substrates toolefins. The olefins can then be separated and purified using knowntechniques.

Post-Production Processing

The olefins produced during fermentation can be separated from thefermentation media. Any known technique for separating olefins fromaqueous media can be used. One exemplary separation process is a twophase (bi-phasic) separation process. This process involves fermentingthe genetically engineered host cells under conditions sufficient toproduce an olefin, allowing the olefin to collect in an organic phase,and separating the organic phase from the aqueous fermentation broth.This method can be practiced in both a batch and continuous fermentationsetting.

Bi-phasic separation uses the relative immiscibility of olefins tofacilitate separation. Immiscible refers to the relative inability of acompound to dissolve in water and is defined by the compound's partitioncoefficient. One of ordinary skill in the art will appreciate that bychoosing a fermentation broth and organic phase, such that the olefinbeing produced has a high log P value, the olefin can separate into theorganic phase, even at very low concentrations, in the fermentationvessel.

The olefins produced by the methods described herein can be relativelyimmiscible in the fermentation broth, as well as in the cytoplasm.Therefore, the olefin can collect in an organic phase eitherintracellularly or extracellularly. The collection of the products inthe organic phase can lessen the impact of the olefin on cellularfunction and can allow the host cell to produce more product.

The methods described herein can result in the production of homogeneouscompounds wherein at least about 60%, 70%, 80%, 90%, or 95% of theolefins produced will have carbon chain lengths that vary by less thanabout 6 carbons, less than about 4 carbons, or less than about 2carbons. These compounds can also be produced with a relatively uniformdegree of saturation. For example, at least about 60%, 70%, 80%, 90%, or95% of the olefins will be monounsaturated, diunsaturated, ortriunsaturated. These compounds can be used directly as fuels, fueladditives, starting materials for production of other chemical compounds(e.g., polymers, surfactants, plastics, textiles, solvents, adhesives,etc.), or personal care additives. These compounds can also be used asfeedstock for subsequent reactions, for example, hydrogenation,catalytic cracking (via hydrogenation, pyrolisis, or both), orepoxidation reactions to make other products.

In some embodiments, the olefins produced using methods described hereincan contain between about 50% and about 90% carbon; between about 5% andabout 25% hydrogen; or between about 5% and about 25% oxygen. In otherembodiments, the olefins produced using methods described herein cancontain between about 65% and about 85% carbon; between about 10% andabout 15% hydrogen; or between about 10% and about 20% oxygen.

Fuel Compositions

The olefins described herein can be used as or converted into a fuel.One of ordinary skill in the art will appreciate that, depending uponthe intended purpose of the fuel, different olefins can be produced andused. For example, a branched olefin may be desirable for automobilefuel that is intended to be used in cold climates. In addition, when theolefins described herein are used as a feedstock for fuel production,one of ordinary skill in the art will appreciate that thecharacteristics of the olefin feedstock will affect the characteristicsof the fuel produced. Hence, the characteristics of the fuel product canbe selected for by producing particular olefins for use as a feedstock.

Using the methods described herein, biofuels having desired fuelqualities can be produced from olefins. Biologically produced olefinsrepresent a new source of biofuels, which can be used as jet fuel,diesel, or gasoline. Some biofuels made using olefins have not beenproduced from renewable sources and are new compositions of matter.These new fuels can be distinguished from fuels derived frompetrochemical carbon on the basis of dual carbon-isotopicfingerprinting. Additionally, the specific source of biosourced carbon(e.g., glucose vs. glycerol) can be determined by dual carbon-isotopicfingerprinting (see, e.g., U.S. Pat. No. 7,169,588, in particular col.4, line 31, to col. 6, line 8).

Hydrocarbons comprising biologically produced hydrocarbons, particularlyα-olefins biologically produced using the fatty acid biosyntheticpathway, have not been produced from renewable sources and, as such, arenew compositions of matter. These new hydrocarbons can be distinguishedfrom hydrocarbons derived from petrochemical carbon on the basis of dualcarbon-isotopic fingerprinting or ¹⁴C dating. Additionally, the specificsource of biosourced carbon (e.g., glucose vs. glycerol) can bedetermined by dual carbon-isotopic fingerprinting (see U.S. Pat. No.7,169,588, which is herein incorporated by reference).

The ability to distinguish biologically produced hydrocarbons frompetroleum based hydrocarbons is beneficial in tracking these materialsin commerce. For example, hydrocarbons or chemicals comprising bothbiologically based and petroleum based carbon isotope profiles may bedistinguished from hydrocarbons and chemicals made only of petroleumbased materials. Hence, the instant materials may be followed incommerce on the basis of their unique carbon isotope profile.

Hydrocarbons can be distinguished from petroleum based fuels bycomparing the stable carbon isotope ratio (¹³C/¹²C) in each fuel. The¹³C/¹²C ratio in a given biologically based material is a consequence ofthe ¹³C/¹²C ratio in atmospheric carbon dioxide at the time the carbondioxide is fixed. It also reflects the precise metabolic pathway.Regional variations also occur. Petroleum, C₃ plants (the broadleaf), C₄plants (the grasses), and marine carbonates all show significantdifferences in ¹³C/¹²C and the corresponding δ¹³C values. Furthermore,lipid matter of C₃ and C₄ plants analyze differently than materialsderived from the carbohydrate components of the same plants as aconsequence of the metabolic pathway.

Within the precision of measurement, ¹³C shows large variations due toisotopic fractionation effects, the most significant of which forbiologically produced hydrocarbons is the photosynthetic mechanism. Themajor cause of differences in the carbon isotope ratio in plants isclosely associated with differences in the pathway of photosyntheticcarbon metabolism in the plants, particularly the reaction occurringduring the primary carboxylation (i.e., the initial fixation ofatmospheric CO₂). Two large classes of vegetation are those thatincorporate the “C₃” (or Calvin-Benson) photosynthetic cycle and thosethat incorporate the “C₄” (or Hatch-Slack) photosynthetic cycle.

In C₃ plants, the primary CO₂ fixation or carboxylation reactioninvolves the enzyme ribulose-1,5-diphosphate carboxylase and the firststable product is a 3-carbon compound. C₃ plants, such as hardwoods andconifers, are dominant in the temperate climate zones.

In C₄ plants, an additional carboxylation reaction involving anotherenzyme, phosphoenol-pyruvate carboxylase, is the primary carboxylationreaction. The first stable carbon compound is a 4-carbon acid which issubsequently decarboxylated. The CO₂ thus released is refixed by the C₃cycle. Examples of C₄ plants are tropical grasses, corn, and sugar cane.

Both C₄ and C₃ plants exhibit a range of ¹³C/¹²C isotopic ratios, buttypical values are about −7 to about −13 per mil for C₄ plants and about−19 to about −27 per mil for C₃ plants (see, e.g., Stuiver et al.,Radiocarbon, 19: 355 (1977)). Coal and petroleum fall generally in thislatter range. The ¹³C measurement scale was originally defined by a zeroset by Pee Dee Belemnite (PDB) limestone, where values are given inparts per thousand deviations from this material. The “δ¹³C”, values arein parts per thousand (per mil), abbreviated, %, and are calculated asfollows:δ¹³C(‰)=[(¹³C/¹²C)_(sample)−(¹³C/¹²C)_(standard)]/(¹³C/¹²C)_(standard)×1000

Since the PDB reference material (RM) has been exhausted, a series ofalternative RMs have been developed in cooperation with the IAEA, USGS,NIST, and other selected international isotope laboratories. Notationsfor the per mil deviations from PDB is δ¹³C. Measurements are made onCO₂ by high precision stable ratio mass spectrometry (IRMS) on molecularions of masses 44, 45, and 46.

The invention provides a hydrocarbon or biofuel produced by any of themethods disclosed herein. Specifically, the hydrocarbon or biofuel canhave a δ¹³C of about −28 or greater, about −27 or greater, −20 orgreater, −18 or greater, −15 or greater, −13 or greater, −10 or greater,and −8 or greater. For example, the hydrocarbon can have a δ¹³C of about−30 to about −15, about −27 to about −19, about −25 to about −21, about−15 to about −5, about −13 to about −7, about −13 to about −10. In someexamples, a biofuel composition can be made that includes an olefinhaving δ¹³C of from about −10.9 to about −15.4, where the olefinaccounts for at least about 85% of biosourced material (i.e., derivedfrom a renewable resource, such as biomass, cellulosic materials, andsugars) in the composition. The invention also provides for ahydrocarbon or biofuel with a δ¹³C of about −10, −11, −12, or −12.3.

Biologically produced hydrocarbons can also be distinguished frompetroleum based hydrocarbons by comparing the amount of ¹⁴C in eachfuel. Because ¹⁴C has a nuclear half life of 5730 years, petroleum basedfuels containing “older” carbon can be distinguished from biofuels whichcontain “newer” carbon (see, e.g., Currie, “Source Apportionment ofAtmospheric Particles,” Characterization of Environmental Particles, J.Buffle and H. P. van Leeuwen, Eds., 1 of Vol. I of the IUPACEnvironmental Analytical Chemistry Series (Lewis Publishers, Inc) (1992)3-74).

The basic assumption in radiocarbon dating is that the constancy of ¹⁴Cconcentration in the atmosphere leads to the constancy of ¹⁴C in livingorganisms. However, because of atmospheric nuclear testing since 1950and the burning of fossil fuel since 1850, ¹⁴C has acquired a second,geochemical time characteristic. Its concentration in atmospheric CO₂,and hence in the living biosphere, approximately doubled at the peak ofnuclear testing, in the mid-1960s. It has since been gradually returningto the steady-state cosmogenic (atmospheric) baseline isotope rate(¹⁴C/¹²C) of about 1.2×10⁻¹², with an approximate relaxation “half-life”of 7-10 years. (This latter half-life must not be taken literally;rather, one must use the detailed atmospheric nuclear input/decayfunction to trace the variation of atmospheric and biospheric ¹⁴C sincethe onset of the nuclear age.)

It is this latter biospheric ¹⁴C time characteristic that holds out thepromise of annual dating of recent biospheric carbon. ¹⁴C can bemeasured by accelerator mass spectrometry (AMS), with results given inunits of “fraction of modern carbon” (f_(M)). f_(M) is defined byNational Institute of Standards and Technology (NIST) Standard ReferenceMaterials (SRMs) 4990B and 4990C. As used herein, “fraction of moderncarbon” or “f_(M)” has the same meaning as defined by National Instituteof Standards and Technology (NIST) Standard Reference Materials (SRMs)4990B and 4990C, known as oxalic acids standards HOxI and HOxII,respectively. The fundamental definition relates to 0.95 times the¹⁴C/¹²C isotope ratio HOxI (referenced to AD 1950). This is roughlyequivalent to decay-corrected pre-Industrial Revolution wood. For thecurrent living biosphere (plant material), f_(M) is approximately 1.1.

The invention provides a hydrocarbon or biofuel which can have an f_(M)¹⁴C of at least about 1. For example, the hydrocarbon or biofuel canhave an f_(M) ¹⁴C of at least about 1.01, an f_(M) ¹⁴C of about 1 toabout 1.5, an f_(M) ¹⁴C of about 1.04 to about 1.18, or an f_(M) ¹⁴C ofabout 1.111 to about 1.124. In some examples, the olefin in the biofuelcomposition can have a fraction of modern carbon (f_(M) ¹⁴C) of, forexample, at least about 1.003, 1.010, or 1.5.

Another measurement of ¹⁴C is known as the percent of modern carbon,pMC. For an archaeologist or geologist using ¹⁴C dates, AD 1950 equals“zero years old”. This also represents 100 pMC. “Bomb carbon” in theatmosphere reached almost twice the normal level in 1963 at the peak ofthermo-nuclear weapons. Its distribution within the atmosphere has beenapproximated since its appearance, showing values that are greater than100 pMC for plants and animals living since AD 1950. It has graduallydecreased over time with today's value being near 107.5 pMC. This meansthat a fresh biomass material, such as corn, would give a ¹⁴C signaturenear 107.5 pMC. Petroleum based compounds will have a pMC value of zero.Combining fossil carbon with present day carbon will result in adilution of the present day pMC content. By presuming 107.5 pMCrepresents the ¹⁴C content of present day biomass materials and 0 pMCrepresents the ¹⁴C content of petroleum based products, the measured pMCvalue for that material will reflect the proportions of the twocomponent types. For example, a material derived 100% from present daysoybeans would give a radiocarbon signature near 107.5 pMC. If thatmaterial was diluted 50% with petroleum based products, it would give aradiocarbon signature of approximately 54 pMC.

A biologically based carbon content is derived by assigning 100% equalto 107.5 pMC and 0% equal to 0 pMC. For example, a sample measuring 99pMC will give an equivalent biologically based carbon content of 93%.This value is referred to as the mean biologically based carbon resultand assumes all the components within the analyzed material originatedeither from present day biological material or petroleum based material.

The invention provides a hydrocarbon or biofuel which can have a pMC ofat least about 50, 60, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, or 100.The invention further provides for a hydrocarbon or fuel which has a pMCof between about 50 to about 100, about 60 to about 100, about 70 toabout 100, about 80 to about 100, about 85 to about 100, and about 87 toabout 98, about 90 to about 95. The invention further provides for ahydrocarbon or biofuel with a pMC of about 90, 91, 92, 93, 94, or 94.2.

Fuel additives are used to enhance the performance of a fuel or engine.For example, fuel additives can be used to alter the freezing/gellingpoint, cloud point, lubricity, viscosity, oxidative stability, ignitionquality, octane level, and/or flash point. In the United States, allfuel additives must be registered with Environmental Protection Agency.The names of fuel additives and the companies that sell the fueladditives are publicly available by contacting the EPA or by viewing theagency's website. One of ordinary skill in the art will appreciate thatthe olefin-based biofuels described herein can be mixed with one or morefuel additives to impart a desired quality.

The olefin-based biofuels described herein can be mixed with otherfuels, such as various alcohols, such as ethanol and butanol, andpetroleum-derived products, such as gasoline, diesel, or jet fuel.

In some examples, the mixture can include at least about 10%, 15%, 20%,30%, 40%, 50%, or 60% by weight of the olefin. In other examples, abiofuel composition can be made that includes at least about 5%, 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90% or 95% of an olefin thatincludes a carbon chain that is 8:1, 10:1, 12:1, 13:1, 14:1, 14:2, 15:1,15:2, 16:1, 16:2, 17:1, 17:2, 18:1, 18:2, 18:3, 19:1, 19:2, 19:3, 20:1,20:2, 20:3, 22:1, 22:2, or 22:3. Such biofuel compositions canadditionally include at least one additive selected from a cloud pointlowering additive that can lower the cloud point to less than about 5°C., or 0° C.; a surfactant; a microemulsion; at least about 5%, 10%,15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, or 95% diesel fuelfrom triglycerides; petroleum-derived gasoline; or diesel fuel frompetroleum.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Identification and Reclassification of a MicroorganismBelonging to the Genus Jeotgalicoccus that is an α-olefin Producer

Micrococcus candicans ATCC 8456 was previously reported to synthesizealiphatic hydrocarbons with carbon chain lengths ranging from C₁₈ to C₂₀(Morrison et al., J. Bacteriol. 108:353-358, 1971). To identify thehydrocarbons produced by this strain, ATCC 8456 cells were cultured in15 mL TSBYE medium (3% Tryptic Soy Broth+0.5% Yeast Extract), for 40-48h at 30° C. Cells from 5 mL of culture were pelleted, resuspended in 1mL methanol, sonicated for 30 min and extracted with 4 mL hexane. Aftersolvent evaporation, samples were resuspended in 0.1 mL hexane andanalyzed by GC-MS. The hydrocarbons were identified as the followingα-olefins: 15-methyl-1-heptadecene (a-C₁₈), 16-methyl-1-heptadecene(i-C₁₈), 1-nonadecene (n-C₁₉), 17-methyl-1-nonadecene (a-C₂₀) and18-methyl-1-nonadecene (i-C₂₀) (i=iso, a=anteiso, n=straight chain)(see, e.g., FIG. 1 and FIG. 2).

Based upon the following analyses, it was determined that ATCC 8456 waspreviously misidentified as belonging to the genus Micrococci. Thephylogenetic classification of ATCC 8456 was reassessed by amplifyingand sequencing the partial 16s rRNA gene using primers Eubac27 and 1492R(see DeLong et al., PNAS 89:5685, 1992). The 16s rRNA sequence ofATCC8456 (SEQ ID NO:3, depicted in FIG. 7C) was analyzed using theclassifier program of the Ribosomal Database Project II. Based upon thisanalysis, the strain was identified as belonging to the genusJeotgalicoccus. The genus Jeotgalicoccus has been previously described(see, e.g., Jung-Hoon et al., Int. J. Syst. Evol. Microbiol. 53:595-602,2003).

Additional analysis using the G+C content of ATCC 8456 was conducted.Jeotgalicoccus is a low G+C Gram-positive bacteria related to the genusStaphylococcus (see FIG. 3). Micrococci are high G+C Gram-positivebacteria. The ends of several clones from a cosmid library of ATCC 8456genomic DNA were sequenced. Based upon a DNA sequence of approximately4,000 bp, the G+C content was determined to be approximately 36%.Nucleotide sequence searches against a non-redundant protein databaserevealed that all sequences with a match to a database entry weresimilar to proteins from low G+C Gram-positive bacteria, such as speciesbelonging to the genus Staphylococcus or Bacillus, but not the genusMicrococcus.

Next, an analysis of the entire genome of ATCC 8456 was conducted. Basedon a DNA sequence of approximately 2.1 MB, the G+C content of the entiregenome was determined to be about 36.7%. In contrast, bacteria of thegenus Micrococcus are known to have high G+C genomes. For example, thegenome of Micrococcus luteus NCTC 2665 has a G+G content of 72.9%(GenBank Accession ABLQ01000001-68). Based upon the G+C contentanalysis, it was determined that the ATCC 8456 microorganism does notbelong to the genus Micrococcus.

Additional Jeotgalicoccus strains were also examined to determine ifthey produced α-olefins. The following strains of Jeotgalicoccus wereexamined: Jeotgalicoccus halotolerans DSMZ 17274, Jeotgalicoccuspsychrophilus DSMZ 19085, and Jeotgalicoccus pinnipedalis DSMZ 17030.Each strain was cultured in 15 mL TSBYE medium (3% Tryptic SoyBroth+0.5% Yeast Extract), and the hydrocarbons were isolated andanalyzed by GC-MS as described above. All three strains producedα-olefins similar to the ones produced by ATCC 8456 (FIGS. 1B, 1C, and1D depict GC-MS traces for the hydrocarbons produced by Jeotgalicoccushalotolerans DSMZ 17274 cells, Jeotgalicoccus pinnipedalis DSMZ 17030cells, and Jeotgalicoccus psychrophilus DSMZ 19085 cells, respectively).These data indicate that the ability to produce α-olefins is widespreadamong the genus Jeotgalicoccus.

Example 2 Production of Increased Levels of Olefins and α-Olefins notNormally Produced by ATCC 8456 Cells Using Fatty Acid Feeding

The fatty acids eicosanoic acid (straight-chain C₂₀ fatty acid),16-methyl octadecanoic acid, and 17-methyl octadecanoic acid(branched-chain C₁₉ fatty acids) were identified as components of ATCC8456's lipids. These fatty acids were deduced to be the directprecursors, after decarboxylation, for 1-nonadecene,15-methyl-1-heptadecene, and 16-methyl-1-heptadecene biosynthesis,respectively. In order to improve α-olefin production and to produceolefins not normally produced by ATCC 8456 cells, fatty acid feedingexperiments were carried out as described below.

ATCC 8456 cells were grown in 15 mL TSBYE medium (3% Tryptic SoyBroth+0.5% Yeast Extract). Fatty acids were added to the culture mediumat final concentrations of 0.5 g/l (0.05%). After growth for 40-48 h at30° C., cells from 5 mL of culture were pelleted, resuspended in 1 mLmethanol, sonicated for 30 min, and extracted with 4 mL hexane. Aftersolvent evaporation, samples were resuspended in 0.1 mL hexane andanalyzed by GC-MS.

When cultures were fed eicosanoic acid, an increase in 1-nonodeceneproduction of approximately 18-fold was observed (see FIG. 4A; blacktraces depict production without fatty acid feeding and gray tracesdepict production with fatty acid feeding). When cultures were fedstearic acid or palmitic acid, an increase in the production of theα-olefins 1-pentadecene and 1-heptadecene, respectively, was observed(see FIG. 4B). These olefins are not normally produced by ATCC 8456cells. This indicated that fatty acids were the direct precursors forα-olefins and that Jeotgalicoccus bacteria could be used toenzymatically convert fatty acids into α-olefins in vivo.

In alternative methods, when resting Jeotgalicoccus cells are fedvarious fatty acids and the production of α-olefins is analyzed, similarresults are observed.

Example 3 In Vitro Synthesis of α-Olefins Using Cell Extracts andPartially Purified Proteins

A cell free extract of ATCC 8456 was used to convert free fatty acidsinto α-olefins. The cell free extract was generated using the followingprocedure: ATCC 8456 cells were grown in TSBYE medium (3% Tryptic SoyBroth+0.5% Yeast Extract) at 30° C. for 24 h with shaking. The cellswere then pelleted from the culture by centrifuging at 3,700 rpm for 20min. The cell pellet was then resuspended in 50 mM Tris buffer pH 7.5with 0.1 M NaCl and 2.0 mM dithiothreitol to a concentration of 0.1 g/mLcells. To this cell slurry, 200 units/mL of lysostaphin (Sigma) wereadded on ice. The cell lysis reaction was allowed to proceed for 30 min.The cells were then sonicated at 12 W on ice for three cycles of 1.5 secof sonication followed by 1.5 sec of rest. Sonication lasted for a totalof 9 sec. This procedure was repeated 5 times with a 1 min intervalbetween each sonication cycle. The lysed cells were then centrifuged at12,000 rpm for 10 min to pellet the cell debris. The supernatant (i.e.,cell free extract) was then removed and used for the conversion of freefatty acids to α-olefins.

After obtaining the cell free extract, the free fatty acids, stearicacid and eicosanoic acid, were converted to α-olefins using the cellfree extract as described below.

First, a 5% stock solution of sodium or potassium stearate was made in1% Tergitol solution (Sigma, St. Louis, Mo.). Next, 6 μL of the stocksolution was added to 1 mL of the cell free extract at room temperatureto obtain a final concentration of 1 mM free fatty acid salt. Thereaction was conducted at room temperature for 3 h. The α-olefins wererecovered by adding 200 μL of ethyl acetate to the mixture, vortexingbriefly, centrifuging briefly, and then removing the organic phase. Theα-olefins were detected using GC/MS.

FIG. 5 shows the GC/MS trace for assaying stearic acid. In sample 1(TIC1; black line in FIG. 5), no stearic acid was added to the cell freeextract. In sample 2 (TIC3; blue line in FIG. 5), the cell free extractwas replaced with 50 mM Tris pH 7.5 buffer with 0.1 M sodium chloride towhich stearic acid was added. In sample 3 (TICS; red line in FIG. 5),stearic acid was added to the cell free extract. The peak at 7.62 minhad the same retention time and the same mass spectra as a 1-heptadecenestandard (Sigma). When eicosanoic acid was added under similarconditions, 1-nonadecene was formed.

Boiling the cell free extract eliminated the production of α-olefinsupon the addition of free fatty acids. This data strongly suggested thatthe ATCC 8456 catalyst was protein based.

The ATCC 8456 cell free extract did not require any additionalco-factors to produce α-olefins. When the cell free extract wassupplemented with several co-factors in 1 mM concentrations, no increasein α-olefin synthesis was observed. The co-factors examined were NAD+,NADP+, NADH, NADPH, FADH₂, SAM, ATP, and CoA. In addition, Mg²⁺ wasexamined at a 10 mM concentration. The co-factor requirement was alsotested by dialyzing the cell free extract with a 10 kDa cut-off membranefor 1.5 h in a volume 200-fold greater than the cell extract volumeusing a dialysis buffer: 50 mM Tris, pH 7.5 with 0.1 M sodium chloride.No decrease in α-olefin synthesis was observed after dialysis.Additionally, no decrease in α-olefin synthesis was observed when 10 mMEDTA pH 7.5 was added to the reaction mixture.

The ATCC 8456 cell free extract was further enriched by carrying out anammonium sulfate precipitation. First, enough ammonium sulfate was addedto the cell free extract to bring the concentration of ammonium sulfateto 50% (wt/vol) saturation. The mixture was stirred gently on ice for 60min and then centrifuged at 13,000 rpm for 30 min. The supernatant wasrecovered and additional ammonium sulfate was added to bring theammonium sulfate concentration to 65% (wt/vol). The mixture was allowedto mix on ice for 60 min and was centrifuged again for 30 min. Thesupernatant was discarded. The pellet was then resuspended in 50 mM Trisbuffer pH 7.5 with 0.1 M sodium chloride. This mixture was then dialyzedin the aforementioned buffer to remove the ammonium sulfate. The cellfree extract treated with ammonium sulfate had the same α-olefinsynthesizing activity as the cell free extract.

Example 4 Purification and Identification of a Protein that ConvertsFatty Acids into α-Olefins

To isolate the protein necessary for α-olefin production from ATCC 8456cells, the following protein purification procedure was carried out.First, 6 L of ATCC 8456 cells were cultured in TSBYE medium at 30° C.for 24 h with shaking. The cells were pelleted at 3,700 rpm for 20 minat 4° C., and the supernatant was discarded. The cell pellet wasresuspended in a solution of 100 mL of 50 mM Tris pH 8.0, 0.1 M NaCl,2.0 mM DTT, and bacterial protease inhibitors. The cell slurry was thenpassed through a french press one time at a pressure of 30,000 psi.Next, the cell slurry was sonicated as described in Example 3 to shearthe DNA. The cell free extract was next centrifuged at 10,000 rpm for 60min at 4° C. The supernatant was then removed and ammonium sulfate wasadded to a final concentration of 50% (wt/vol) ammonium sulfatesaturation. The mixture was gently stirred at 4° C. for 60 min and thencentrifuged at 10,000 rpm for 30 min. The supernatant was then removedand additional ammonium sulfate was added to 65% (wt/vol) saturation.The mixture was stirred again for 60 min at 4° C. and centrifuged at10,000 rpm for 30 min. The supernatant was discarded. The remainingpellet was resuspended in 50 mL of 50 mM Tris pH 8.0 and 2.0 mM DTT.

The mixture was passed through a 5 mL HiTrap SP column (GE Healthcare)at 3 mL/min and 4° C. The following buffers were used as an elutiongradient: buffer A contained 50 mM Tris pH 8.0 and 2.0 mM DTT and bufferB contained 50 mM Tris pH 8.0, 1.0 M NaCl, and 2.0 mM DTT. After thecolumn was loaded with the mixture, the column was washed with 40%buffer B. Next a 20 min gradient of 40% buffer B to 100% buffer B at 3.0mL/min was carried out. 5 mL fractions were collected during the elutiongradient. Each fraction was tested for activity as described in Example3. Fractions containing α-olefin production activity typically elutedbetween 600 and 750 mM NaCl concentration. Fractions containing activitywere then pooled and dialyzed into buffer A.

The dialyzed protein fraction was then loaded onto a 1 mL ResourceQ (GEHealthcare) column at 4 mL/min at 4° C. Buffer B used with the HiTrap SPcolumn was used for the ResourceQ column. A 7-min elution gradientbetween 0% buffer B and 25% buffer B was run at 4 mL/min. 1.5 mLfractions were collected and assayed for activity. Active fractionseluted between 150 and 200 mM NaCl concentrations. Fractions containingactivity were then pooled and concentrated with a Millipore Amiconprotein concentrator (4 mL and 10 kDa exclusion size) to about 50 μL.The approximate protein concentration was determined with a Bradfordassay (Biorad). Final protein concentrations ranged from about 5 mg/mLto about 10 mg/mL. 30 μL of protein was then loaded onto a SDS PAGE gel(Invitrogen) along with an appropriate protein molecular weight marker.The gel was stained with Simple Safe Coomassie stain (Invitrogen). FIG.6 depicts a representative gel. Two intense protein bands at 50 kDa and20 kDa were observed.

To determine the identity of the protein bands, the bands were excisedfrom the gel, digested with trypsin, and analyzed using LC/MS/MS. TheLC/MS/MS data was analyzed using the program Mascot (Mann et al., Anal.Chem. 66:4390-4399, 1994). The ATCC 8456 genome was sequenced. Thegenomic data was used to interpret the LC/MS/MS data and to determinethe identity of the protein bands. The 50 kDa band had a strong matchwith ORF880. The Mascot score assigned to this match was 919, a highscore. Furthermore, ORF880 has a predicted molecular weight of 48,367Da. The nucleotide and amino acid sequences of orf880 are presented inFIGS. 7A (SEQ ID NO:1) and 7B (SEQ ID NO:2), respectively.

Example 5 Heterologous Expression of Jeotgalicoccus ATCC 8456 orf880 inE. coli

Jeotgalicoccus ATCC 8456 ORF880 was identified as one of the two majorproteins in a highly purified enzyme fraction that catalyzed theconversion of free fatty acids to α-olefins. The genomic DNA encodingATCC 8456_orf880 was cloned into pCDF-Duet1 under the control of the T7promoter, and E. coli was transformed with various vectors, as describedbelow. The E. coli cells were grown, and the hydrocarbons produced bythe cells were analyzed as described in Example 2. When 0.05% stearicacid was fed to cultures of E. coli transformed with the8456_orf880-containing vector, the expression of 8456_orf880 led to theformation of 1-heptadecene in E. coli (see FIG. 8, depicting GC/MStraces of α-olefins from E. coli either without (black) or with (gray)expression of 8456_orf880). In contrast, adding 0.05% stearic acid tocultures of E. coli transformed with a vector control (not containingATCC— orf880) did not result in the production of 1-heptadecene. Thisdemonstrated that 8456_ORF880 synthesized α-olefins from free fattyacids in an E. coli heterologous host. This result indicates thatα-olefin biosynthesis can be performed in heterologous organisms.Additionally, when E. coli cells expressing 8456_orf880 protein were fedwith 0.05% palmitic acid or 0.05% eicosanoic acid, the production of1-pentadecene or 1-nonadecene, respectively, was observed.

Example 6 Production of α-Olefins from Glucose by HeterologousExpression of Jeotgalicoccus ATCC 8456 orf880 in E. coli MG1655 ΔfadDConstruction of fadD Deletion Strain

The fadD gene of E. coli MG1655 was deleted using the lambda red system(Datsenko et al., 2000, Proc. Natl. Acad. Sci. USA. 97: 6640-6645) asdescribed below.

The chloramphenicol acetyltransferase gene from pKD3 was amplified withthe primers fad1:

(5′-TAACCGGCGTCTGACGACTGACTTAACGCTCAGGCTTTATTGTCCACTTTGTGTAGGCTGGAGCTGCTTCG-3′),and fad2:

(5′-CATTTGGGGTTGCGATGACGACGAACACGCATTTTAGAGGTGAAGAATTGCATATGAATATCCTCCTTTAGTTCC-3′).

This PCR product was electroporated into E. coli MG1655 (pKD46). Thecells were plated on L-chloramphenicol (30 μg/mL)(L-Cm) and grownovernight at 37° C. Individual colonies were picked on to another L-Cmplate and grown at 42° C. These colonies were then patched to L-Cm andL-carbenicillin (100 mg/mL) (L-Cb) plates and grown at 37° C. overnight.Colonies that were Cm^(R) and Cb^(S) were evaluated further by PCR toensure the PCR product inserted at the correct site. PCR verificationwas performed on colony lysates of these bacteria using the primers fadF(5′-CGTCCGTGGTAATCATTTGG-3′) and fadR (5′-TCGCAACCTTTTCGTTGG-3′). Theexpected size of the ΔfadD::Cm deletion was about 1200 bp (FIG. 11). Thechloramphenicol resistance gene was eliminated using a FLP helperplasmid as described in Datsenko et al. (2000) Proc. Natl. Acad. Sci.USA. 97: 6640-6645. PCR verification of the deletion was performed withprimers fadF and fadR (FIG. 11). The MG1655 ΔfadD strain was unable togrow on M9+oleate agar plates (oleate as carbon source). It was alsounable to grow in M9+oleate liquid media.

Expression of Jeotgalicoccus ATCC 8456_orf880 in E. coli MG1655 ΔfadD

The genomic DNA encoding ATCC 8456_orf880, which was codon-optimized forexpression in E. coli (SEQ ID NO:4), was cloned into vector OP80(pCL1920 derivative) under the control of the P_(trc) promoter, and E.coli MG1655 ΔfadD was transformed with the resulting vector. The E. colicells were grown at 37° C. in M9 mineral medium supplemented with 20μg/mL uracil and 100 μg/mL spectinomycin. Glucose (1%, w/v) was the onlysource of carbon and energy. When the culture reached an OD₆₀₀ of 0.8 to1.0, IPTG (1 mM) and 0.5 mM delta-aminolaevulinic acid (hemebiosynthesis precursor) were added. The temperature was shifted to 25°C. After growth for an additional 18 to 24 h at 25° C., the cells from10 mL of culture were pelleted, resuspended in 1 mL methanol, sonicatedfor 30 min, and extracted with 4 mL hexane. After solvent evaporation,the samples were resuspended in 0.1 mL hexane and analyzed by GC-MS. Incontrast to the vector-only control, E. coli cells transformed with theorf880-bearing vector produced the α-olefins: 1-pentadecene andheptadecadiene (see FIG. 12). This result indicates that expression oforf880 confers on E. coli the ability to biosynthesize α-olefins whengrown on glucose and that the direct precursors are the most abundantfatty acids in E. coli, namely hexadecanoic acid and vaccenic acid(11-cis-octadecenoic acid).

Example 7 Production of α-Olefins in E. coli by Heterologous Expressionof Corynebacterium efficiens YS-134 orfCE2459

Genomic DNA encoding C. efficiens YS-134 (DSMZ44549) orfCE2459 (SEQ IDNO:6) was cloned into the NcoI and EcoRI sites of vector OP-80 (pCL1920derivative) under the control of the P_(trc) promoter. The resultingconstruct was then transformed into E. coli C41 ΔfadE.

The E. coli C41 ΔfadE was constructed using primers YafV_NotI andIvry_O1 to amplify about 830 bp upstream of fadE and primers Lpcaf_oland LpcaR_Bam to amplify about 960 bp downstream of fadE. Overlap PCRwas used to create a construct for in frame deletion of the completefadE gene. The fadE deletion construct was cloned into thetemperature-sensitive plasmid pKOV3, which contained a sacB gene forcounterselection, and a chromosomal deletion of fadE was made accordingto the method of Link et al., J. Bact. 179:6228-6237, 1997. This strainhas the acyl-CoA dehydrogenase gene (fadE) deleted, therefore it can notdegrade activated fatty acids.

The cells were grown at 37° C. in M9 minimal media supplemented with 20μg/mL uracil, 1% glucose (w/v), and 100 μg/mL spectinomycin. When theculture reached an OD₆₀₀ of 0.8 to 1.0, it was induced with 1 mM IPTGsupplemented with 0.5 mM delta-aminolaevulinic acid (heme biosynthesisprecursor), and 0.05% stearic acid (as a substrate for orfCE2459). Thetemperature was shifted to 25° C. and cells were allowed to grow for anadditional 18-20 h Cells from 1 mL of culture were resuspended with 100μL of methanol, sonicated for 30 min, and extracted with 300 μL of ethylacetate. After vortexing for 15 min, 300 μL of water was added, and themixture was vortexed for another 15 min before centrifugation at 15,000rpm for 10 min. The solvent layer was analyzed by GC-MS.

As shown in FIG. 13, E. coli cells transformed with an orfCE2459-bearingvector produced 1-heptadecadiene without stearic acid feeding andproduced 1-heptadecene with stearic acid feeding. This result indicatesthat expression of orfCE2459 confers on E. coli the ability tobiosynthesize α-olefins (e.g., 1-heptadecadiene) when grown on glucose(FIG. 13B) and that orfCE2459 can convert fed fatty acids, such asstearic acid, into α-olefins, such as 1-heptadecene (FIG. 13C).

Example 8 Production of α-Olefins in E. coli by Heterologous Expressionof Kokuria rhizophila orfKRH21570

Genomic DNA encoding Kokuria rhizophila DC2201 orfKRH21570 (SEQ IDNO:8), which was codon optimized (SEQ ID NO:9) for expression in E.coli, was cloned into the NdeI and XhoI sites of vector OP-183 (pACYC177derivative) under the control of the P_(trc) promoter. The resultingconstruct was then transformed into E. coli C41 ΔfadE. The cells weregrown, extracted, and analyzed as in described in Example 7.

As shown in FIG. 14, E. coli cells transformed with codon optimizedorfKRH21570-bearing vector produced 1-heptadecadiene without stearicacid feeding and produced 1-heptadecene with stearic acid feeding. Thisresult indicates that expression of orfKRH21570 confers on E. coli theability to biosynthesize α-olefins (e.g., 1-heptadecadiene) when grownon glucose (FIG. 14C) and that orfKRH21570 can convert fed fatty acids,such as stearic acid, into α-olefins, such as 1-heptadecene (FIG. 14D).

Example 9 Production of α-Olefins in Bacillus subtilis by HeterologousExpression of Jeotgalicoccus sp. ATCC8456 orf880

Genomic DNA encoding Jeotgalicoccus ATCC 8456_orf880 was cloned into theBamHI and XbaI sites of vector pHT01 under the control of the P_(grac)promoter. B. subtilis IHA01 (lacA::spec leuB8 metB5 r(−)m(+) Sp,obtained from Bacillus Genetic Stock Center, Columbus, Ohio, strainnumber BGSC 1A785) was transformed with the resulting vector. The B.subtilis cells were grown at 37° C. in minimal medium supplemented with2% glucose, 0.3 mM leucine, 0.3 mM methionine, and 10 mg/Lchloramphenicol. When the cultures reached an OD₆₀₀ of 0.8 to 1.0, 1 mMIPTG was added (to induce expression of orf880) along with 0.5 mM5-amino laevulinic acid (as a precursor for heme biosynthesis) and 0.05%stearic acid (as a substrate for ORF880). The temperature was shifted to25° C. and after growth for additional 18-24 h at 25° C., cells from a 1mL culture were pelleted, resuspended in 100 μL methanol, sonicated for1 h, and extracted with 300 μL ethyl acetate. 300 μL of water was addedbefore separating the organic layer by centrifugation at 15,000 rpm for5 min. The solvent layer was analyzed by GC-MS.

In contrast to cells transformed with the vector only control, B.subtilis cells transformed with Jeotgalicoccus sp. ATCC8456orf880-bearing vector produced 1-heptadecene from stearic acid (see FIG.15B). This example demonstrates that in B. subtilis fatty acids, such asstearic acid, can be converted to α-olefins by heterologous expressionof Jeotgalicoccus ATCC 8456_orf880 in Bacillus subtilis.

Example 10 Production of α-Olefins in Bacillus subtilis by HeterologousExpression of Corynebacterium efficiens YS-134 orfCE2459

Genomic DNA encoding Corynebacterium efficiens YS-134 orfCE2459 wascloned into the BamHI and XbaI sites of vector pHT01 under the controlof the P_(grac) promoter and transformed into B. subtilis IHA01 cells.The resulting cells were grown, extracted, and analyzed as described inExample 9.

In contrast to cells transformed with the vector only control, B.subtilis cells transformed with Corynebacterium efficiens YS-134orf_CE2459-bearing vector produced 1-heptadecene from stearic acid (seeFIG. 15). This demonstrates that in B. subtilis fatty acids, such asstearic acid, can be converted to α-olefins by heterologous expressionof C. efficiens orf_CE2459.

Example 11 Production of α-Olefins in Saccharomyces cerevisiae byHeterologous Expression of Jeotgalicoccus sp. ATCC8456 orf880

Genomic DNA encoding Jeotgalicoccus sp. ATCC 8456_orf880 is cloned intothe ApaI and XhoI sites of the yeast expression vector pESC-His. S.cerevisiae (BY4741) yeast cells are then transformed with either aplasmid containing orf880 or a plasmid not containing orf880. Thetransformed yeast cells are cultured under suitable conditions to allowexpression of ORF880. The cells are pelleted and then lysed usingYeastBuster™ (Novagen, Madison, Wis.). The α-olefins are extracted andanalyzed as described in Examples 6 and 7.

Jeotgalicoccus sp. ATCC8456_orf880 expressed in S. cerevisiae producesα-olefins. Similar methods are used to express Jeotgalicoccus sp.ATCC8456_orf880 in any cell of interest, after which cell lysates areprepared and the production of α-olefins analyzed.

Example 12 In Vitro Synthesis of α-Olefins by Jeotgalicoccus sp.ATCC8456 orf880 Heterologously Expressed in and Purified from E. coli

Expression and Purification of Jeotgalicoccus sp. ATCC8456_orf880

The genomic DNA encoding Jeotgalicoccus sp. ATCC8456_orf880 was clonedinto the NdeI and XhoI sites of vector pET15b (Novagen) under thecontrol of the T7 promoter for expression in and purification from E.coli. This plasmid expressed an N-terminal His-tagged version of8456_orf880.

An E. coli BL21 strain (DE3) (Invitrogen) was transformed withpET15b-orf880 using routine chemical transformation techniques. Proteinexpression was carried out by first inoculating a colony of the E. colistrain in 5 mL of LB media supplemented with 100 mg/L of carbenecillinand shaken overnight at 37° C. to produce a starter culture. Thisstarter culture was used to inoculate 1 L of LB media supplemented with100 mg/L of carbenecillin. The culture was shaken at 37° C. until anOD₆₀₀ value of 0.6 was reached. The culture was placed on ice for 10 minbefore IPTG was added to a final concentration of 250 μM. The culturewas then shaken at 18° C. for approximately 18 h. The culture was thencentrifuged at 3,700 rpm for 20 min at 4° C. The pellet was thenresuspended in 30 mL of buffer containing 100 mM sodium phosphate bufferat pH 7.2 supplemented with Bacterial ProteaseArrest (GBiosciences). Thecells were then sonicated at 12 W on ice for 9 sec with 1.5 sec ofsonication followed by 1.5 sec of rest. This procedure was repeated 5times with one min intervals between each sonication cycle. The cellfree extract was centrifuged at 10,000 rpm for 30 min at 4° C. 5 mL ofNi-NTA (Qiagen) was added to the supernatant and the mixture was gentlystirred at 4° C. The slurry was passed over a column removing the resinfrom the lysate. The resin was then washed with 30 mL of buffercontaining 100 mM sodium phosphate buffer at pH 7.2 plus 30 mMimidazole. Finally, the protein was eluted with 15 mL of 100 mM sodiumphosphate buffer at pH 7.2 plus 250 mM imidazole. The protein solutionwas dialyzed with 200 volumes of 100 mM sodium phosphate buffer at pH7.2. Protein concentration was determined using the Bradford assay(Biorad). 125 μg/mL of protein was obtained.

In Vitro Synthesis of α-Olefins from Fatty Acid Substrates

In order to assay the in vitro fatty acid substrate specificity ofORF880, potassium salts of the following fatty acids were prepared:tetradecanoic acid, hexadecanoic acid, octadecanoic acid, eicosanoicacid, and behenic acid (Sigma). The fatty acid solutions were made with2% ethanol and 2% Tergitol solution (Sigma, St. Louis, Mo.) to a finalconcentration of 20 mM.

The kinetics of the decarboxylation reaction was determined usingpotassium octadecanoate. A 200 μL reaction was prepared with thefollowing reactant concentrations: 1.25 μM of ORF880, 200 μM ofpotassium octadecanoate, 200 μL dithiothreitol, and 100 mM sodiumphosphate buffer at pH 7.2. The reactions were incubated at roomtemperature and time points were taken in duplicate between 5 min and120 min. Reactions were quenched and extracted by adding 100 μL of ethylacetate containing 1-octadecene at 5 mg/L as an internal reference.Samples were analyzed using GC/MS using the alkane-1 splitless method,which is performed using the following parameters: run time: 20 min;column: HP-5-MS Part No. 19091S-433E (length of 30 meters; I.D.: 0.25 mmnarrowbore; film: 0.25 μM); sample: standard ethyl acetate extraction;inject: 1 μL Agilent 6850 inlet; inlet: 300° C. splitless; carrier gas:helium; flow: 1.3 mL/min; oven temp: 100° C. hold 5 min, 320 at 20°C./min, 320 hold 5 min; det: Agilent 5975B VL MSD; det. temp: 300° C.;scan: 50-500 M/Z. Calibration curves were generated using 1-heptadecenedissolved in ethyl acetate. Based upon this analysis, the productproduction was determined to be linear from 5 min to 60 min.

To assay the reaction rates of different fatty acid substrates, thefollowing reactions were prepared at 200 μL scales: 1.0 μM ORF880enzyme, 200 μM of a test fatty acid salt, 200 μL dithiothreitol, and 100mM sodium phosphate buffer at pH 7.2. The reactions were carried out atroom temperature and time points were taken in triplicate at 20 min and47 min using the extraction and analysis procedure described above.Reference curves were generated using available chemical standards. Insome instances, the chemical standards were not available (for example,cis-9-heneicosene was used as a reference for 1-heneicosene and9-tricosene was used as a reference for 1-tricosene). Activities werecalculated by taking the difference between the average α-olefinconcentrations for each substrate at 47 min and 20 min and then dividingthe difference by 27 min. The results are summarized in Table 7.

TABLE 7 Activity of ORF880 with different fatty acid substrates Activity(nM alkene Substrate produced/min) tetradecanoic acid 22.9 hexadecanoicacid 181.9 octadecanoic acid 77.2 eicosanoic acid 19.7 behenic acid 30.6These results demonstrate that heterologously expressed ORF880 was ableto convert various fatty acid substrates to α-olefins in vitro. The dataalso show that ORF880 had highest activity when hexadecanoic acid wasthe fatty acid substrate.

Example 13 In Vitro Synthesis of α-Olefins by Corynebacterium efficiensYS-134 orfCE2459 Heterologously Expressed in E. coli

An E. coli BL21 strain (DE3) (Invitrogen) was transformed with pET15-orfCE2459 using routine transformation techniques. Protein expression wascarried out by first inoculating a colony of the E. coli strain in 5 mLof LB media supplemented with 100 mg/L of carbenecillin and then shakingovernight at 37° C. to produce a starter culture. This starter culturewas used to inoculate 0.5 L of LB media supplemented with 100 mg/L ofcarbenecillin. The culture was shaken at 37° C. until an OD₆₀₀ value of0.6 was reached. The culture was placed on ice for 10 min before IPTGwas added to a final concentration of 250 μM. δ-Aminolevulinic acid wasalso added to a final concentration of 0.5 mM. The culture was thenshaken at 25° C. for approximately 18 h. The culture was thencentrifuged at 3,700 rpm for 20 min at 4° C. The pellet was thenresuspended in 7 mL of buffer containing 100 mM sodium phosphate bufferat pH 7.2 supplemented with Bacterial ProteaseArrest (GBiosciences). Thecells were then sonicated at 12 W on ice for 9 sec with 1.5 sec ofsonication followed by 1.5 sec of rest. This procedure was repeated 4times with one min intervals between each sonication cycle. The cellfree extract was centrifuged at 10,000 rpm for 30 min at 4° C.

To assay the activity of ORF CE2459, 200 μL of lysate was incubated with200 μM of hydrogen peroxide and 200 μM of potassium octadecanoate. As anegative control, the same reaction was set up, but without the additionof either cell lysate (100 mM sodium phosphate pH 7.2 was used in itsplace) or free fatty acid. The reactions were incubated at roomtemperature overnight. The reactions were then extracted with 100 μL ofethyl acetate with 1-octadecene added at 5 mg/L as an internal standard.Samples were analyzed using GC/MS using the alkane-1 splitless methodwhich was performed using the following parameters: run time: 20 min;column: HP-5-MS Part No. 19091S-433E (length of 30 meters; I.D.: 0.25 mmnarrowbore; film: 0.25 μM); sample: standard ethyl acetate extraction;inject: 1 μL Agilent 6850 inlet; inlet: 300° C. splitless; carrier gas:helium; flow: 1.3 mL/min; oven temp: 100° C. hold 5 min, 320 at 20°C./min, 320 hold 5 min; det: Agilent 5975B VL MSD; det. temp: 300° C.;scan: 50-500 M/Z.

As shown in FIG. 17, an E. coli lysate expressing orfCE2459 was capableof converting octadecanoic acid into 1-heptadecene. The peak at 5.20 mincorresponds to 1-heptadecene and the peak at 8.76 is the internalstandard 1-octadecene. Peak identification was determined by comparingthe retention time and the MS fragmentation pattern with authenticreferences from Sigma. This result demonstrates that ORF CE2459 exhibitssimilar in vitro fatty acid decarboxylase activity as ORF880.

Example 14 In Vitro Synthesis of α-Olefins by Kokuria rhizophilaorf_KRH21570 Heterologously Expressed in and Purified from E. coli

The genomic DNA encoding Kokuria rhizophila orf_KRH21570 was cloned intothe NdeI and XhoI sites of vector pET15b (Novagen) under the control ofthe T7 promoter for expression in and purification from E. coli. Thisplasmid expressed an N-terminal His-tagged

E. coli C41 DE3 (ΔfadE) cells were transformed with pET15b-orfKRH21570using routine transformation techniques. Protein expression was carriedout by first inoculating 5 mL of LB broth supplemented with 100 mg/Lcarbenicillin with a colony of the E. coli strain which was followed byshaking the cells overnight at 37° C. to produce a starter culture. Thisstarter culture was used to inoculate 0.5 L of LB media supplementedwith 100 mg/L of carbenicillin. The culture was shaken at 37° C. untilan OD₆₀₀ value of 0.6 was reached, after which it was induced with 1 mMIPTG and 0.5 mM delta-aminolaevulinic acid. The culture was then shakenat 25° C. for approximately 18 h. The protein was purified as describedin Example 12.

To assay the activity of ORF_KRH21570, 200 μL of purified protein wasincubated with 200 μM of hydrogen peroxide and 200 μM of potassiumhexadecanoate. As a negative control, the same reaction was conducted,but without the addition of either purified protein (100 mM sodiumphosphate pH 7.2 was used in its place) or free fatty acid. Thereactions were performed in duplicates and incubated at room temperatureovernight. The reactions were then extracted with 100 μL of ethylacetate with 1-octadecene added at 5 mg/L as an internal standard.Trimethylanilinium hydroxide was added to the ethyl acetate layer at a1:1 ratio and the extracts analyzed by GC/MS using theMAR_splitless_short method. The parameters used were as follows: 9.50min; column: DB5-ht Part No. J&W 122-5711 (length of 15 meters; I.D.:0.25 mm narrowbore; film: 0.10 μM); sample: standard ethyl acetateextraction; inject: 1 μL Agilent 6850 inlet; inlet: 300 C splitless;carrier gas: helium; flow: 1.3 mL/min; oven temp: 80° C. hold 1.0 min,320 at 30° C./min, 320 hold 0.5 min; det: Agilent 5975B VL MSD; E1 mode;det. temp: 230° C.; scan: 50-330 M/Z.

As shown in FIG. 18, Kokuria rhizophila ORF_KRH21570 was capable ofconverting hexadecanoic acid to 1-pentadecene in vitro.

Example 15 In Vitro Synthesis of α-Olefins by Methylobacterium populiorf_Mpop1292 Heterologously Expressed in and Purified from E. coli

The genomic DNA encoding Methylobacterium populi orf_Mpop1292 (SEQ IDNO:11) was cloned into the NdeI and XhoI sites of vector pET15b(Novagen) under the control of the T7 promoter for expression in andpurification from E. coli. This plasmid expressed an N-terminalHis-tagged version of orf_KRH21570.

E. coli C41 DE3 (ΔfadE) cells were transformed with pET15b-orf_Mpop1292using routine transformation techniques. Protein expression was carriedout by first inoculating 5 mL of LB broth supplemented with 100 mg/Lcarbenicillin with a colony of the E. coli strain, and then shaking thecells overnight at 37° C. to produce a starter culture. This starterculture was used to inoculate 0.5 L of LB media supplemented with 100mg/L of carbenicillin. The culture was shaken at 37° C. until an OD₆₀₀value of 0.6 was reached, after which it was induced with 1 mM IPTG and0.5 mM delta-aminolaevulinic acid. The culture was then shaken at 25° C.for about 18 h. The protein was purified as described in Example 12 andassayed as described in Example 14.

As shown in FIG. 19, Methylobacterium populi ORF_Mpop1292 was capable ofconverting hexadecanoic acid to 1-pentadecene in vitro.

Example 16 Production of α-Olefins in E. coli Through HeterologousExpression of Bacillus subtilis Fatty Acid Hydroxylase, P450_(Bsβ)

The genomic DNA encoding Bacillus subtilis fatty acid hydroxylase,Cyp152A1 (SEQ ID NO:13) or P450_(Bsβ), was cloned into the NdeI and XhoIsites of vector OP-183 (pACYC177 derivative) under the control of theP_(trc) promoter. The construct was used as a template to introduce amutation at position 85 changing the Glu to His using the followingprimer: GTTAATGCGATTCAcGGAATGGATGGC. The resulting constructs were thentransformed into E. coli C41 (ΔfadE). The cells were grown at 37° C. inM9 minimal media supplemented with 20 μg/mL uracil, 1% glucose (w/v),and 100 μg/mL carbenicillin. When the culture reached OD₆₀₀ of 0.8-1.0,it was induced with 1 mM IPTG; supplemented with 0.5 mMdelta-aminolaevulinic acid (heme biosynthesis precursor) and 0.05%stearic acid (as a substrate for Bs_(b)). The temperature was shifteddown to 25° C. and cells were allowed to grow for an additional 18-20 h.Cells from 1 mL of culture was resuspended with 100 μL of methanol,sonicated for 30 min, and extracted with 300 μL of ethyl acetate. Aftervortexing the extract for 15 min, 300 μL of water was added, vortexedfor another 15 minutes before centrifugation at 15,000 rpm for 10minutes. The solvent layer was analyzed by GC-MS.

As shown in FIG. 20, E. coli cells transformed with Bacillus subtilisP450_(Bsβ)-bearing vectors produced 1-heptadecene with stearic acidfeeding. This result indicates that Bacillus subtilis P450_(Bsβ) canconvert fed fatty acids, such as stearic acid, into α-olefins, such as1-heptadecene (FIG. 20). Both the wildtype protein and theGln85-His-mutant protein were capable of carrying out this reaction.

Example 17 Specific Activity of α-Olefin Production and α and β HydroxyFatty Acid Production Using ORF880, CYP152A1 (P450_(Bsβ)), and CYP152A1Q85H

The specific activity of α-olefin formation as well as α-(2) andβ-(3)hydroxy fatty acid formation were determined for ORF880, CYP152A1,and CYP152A1 Q85H (see Examples 12 and 16). An E. coli BL21 strain (DE3)(Invitrogen) was transformed with either pET15b-orf 880,pET15b-cyp152A1, and pET15b-cyp152A1 Q85H using routine chemicaltransformation techniques. Protein expression was carried out by firstinoculating a colony of the E. coli strain in 5 mL of LB mediasupplemented with 100 mg/L of carbenicillin and shaken overnight at 37°C. to produce a starter culture. This starter culture was used toinoculate 0.5 L of LB media supplemented with 100 mg/L of carbenicillin.The culture was shaken at 37° C. until an OD₆₀₀ value of 0.6 wasreached. The culture was placed on ice for 10 min before IPTG was addedto a final concentration of 250 μM. The culture was shaken at 25° C. forapproximately 18 h, and then centrifuged at 3,700 rpm for 20 min at 4°C. The pellet was then resuspended in 10 mL of buffer containing 100 mMsodium phosphate buffer at pH 7.2 supplemented with BacterialProteaseArrest (GBiosciences). The cells were then sonicated at 12 W onice for 9 sec with 1.5 sec of sonication followed by 1.5 sec of rest.This procedure was repeated 5 times with one min intervals between eachsonication cycle. The cell free extract was centrifuged at 10,000 rpmfor 30 min at 4° C. 5 mL of Ni-NTA (Qiagen) was added to thesupernatant, and the mixture was gently stirred at 4° C. The slurry waspassed over a column removing the resin from the lysate. The resin wasthen washed with 30 mL of buffer containing 100 mM sodium phosphatebuffer at pH 7.2 plus 30 mM imidazole. Finally, the protein was elutedwith 15 mL of 100 mM sodium phosphate buffer at pH 7.2 plus 250 mMimidazole. The protein solution was dialyzed with 200 volumes of 100 mMsodium phosphate buffer at pH 7.2 with 20% glycerol. Proteinconcentration was determined using the Bradford assay (Biorad). 578μg/mL of ORF880, 865 μg/mL of CYP152A1, and 653 μg/mL of CYP152A1 Q85Hprotein was obtained.

To determine the specific activity with potassium hexadecanoate, thefollowing reactions were set-up where each time point consisted of 500μL. Each reaction contained of 500 μM H₂O₂, 200 μM of potassiumhexadecanoate, 100 mM sodium phosphate buffer at pH 7.2, and protein atthe following concentrations: ORF880-0.23 or 0.46 μM; CYP152A1-8.65,17.3, or 34.6 μM; CYP152A1 Q85H-6.53, 13.06, or 26.12 μM. The reactionswere carried out at room temperature and time points were taken at 0, 1,3, 5, 7, 10, 14, and 20 minutes. Reactions were quenched with 50 μL of12 M HCl and then extracted with 200 μL of ethyl acetate. 50 μL of thisextract was then reacted with 50 μLN,O-Bis(trimethylsilyl)trifluoroacetamide with trimethyl-chlorosilane(Aldrich). Samples were analyzed using GC/MS using the followingparameters: run time: 20 min; column: HP-5-MS Part No. 190915-433E(length of 30 meters; I.D.: 0.25 mm narrowbore; film: 0.25 μM); inject:1 μL Agilent 6850 inlet; inlet: 300° C. splitless; carrier gas: helium;flow: 1.3 mL/min; oven temp: 100° C. hold 5 min, 320° C. at 20° C./min,320° C. hold 5 min; det: Agilent 5975B VL MSD; det. temp: 300° C.; scan:50-500 M/Z. Calibration curves were generated using 1-pentadecene,α(2)-hydroxyhexadecanoic acid, and β(3)-hydroxyhexadecanoic aciddissolved in ethyl acetate and derivatized as described herein.

Based upon this analysis, product formation was determined to be linearwithin the first three minutes. The specific activities for each enzymeconcentration in the linear range were averaged for a given reaction(e.g., decarboxylation, α-hydroxylation, or β-hydroxylation). FIG. 21summarizes the results. The specific activity for 1-pentadeceneformation was similar for ORF880 and CYP152A1. Mutating the glutamine 85residue to a histidine increased the specific activity of 1-pentadeceneformation (i.e., increased fatty acid decarboxylase activity). Thismutation also increased the rate of β-hydroxyhexadecanoic acid formationand lowered the rate of α-hydroxyhexadecanoic acid formation as comparedwith the wild-type version of CYP152A1. The rate of hydroxyhexadecanoicacid production by ORF880 was about 1/50^(th) of the rates exhibited bythe two CYP152A1 enzymes. The ratio of 1-pentadecene formation (i.e.decarboxylation) to hydroxyhexadeconic acid formation (i.e.,hydroxylation) activities for each enzyme is given in FIG. 22.

Example 18 Analysis of ORF880 Homolog Activity

As described in Examples 7 through 17, five P450 enzymes were able toconvert fatty acids into α-olefins in-vivo and in-vitro, and three ofthese P450 enzymes were also able to convert fatty acids into β-hydroxyfatty acids (see Table 8). In contrast, two P450 enzymes, Bacillusclaussii orf_ABC3040 and Sphingomonas paucimobilis P450_(spα), were notable to make α-olefins or β-hydroxy fatty acids. These P450 enzymesconverted fatty acids only to the corresponding α-hydroxy fatty acids(see Table 8). Consequently, there appears to be a correlation betweenthe ability of a P450 enzyme to hydroxylate fatty acids in theβ-position and to decarboxylate fatty acids to the α-olefin, and theinability of a P450 enzyme to decarboxylate fatty acids to α-olefins ifthe enzyme can only hydroxylate fatty acids in the α-position. In otherwords, P450 enzymes that hydroxylate fatty acids in the β-position alsohave the potential to decarboxylate these fatty acids to α-olefins.

Table 8 also demonstrates that when a His or Met is at position 85, theenzyme has the ability to produce α-olefins. Table 8 also shows thepercent identity of each enzyme tested relative to Jeotgalicoccus sp.8456 ORF880.

TABLE 8 ORF880 Homologs Tested Position Percent Makes Makes MakesOrganism/P450 enzyme 85 ³ Identity α-olefin ⁴ α-hyFA ⁴ β-hyFA ⁴Jeotgalicoccus sp. 8456 orf880 His 100 + − + (fatty acid decarboxylase)Corynebacterium efficiens NP_739069 His 27 + (−) (−) (orf_CE2459) ¹Kokuria rhizophila YP_001856010 His 29 + − + (orf_KRH21570) Bacillusclausii YP_176535 Gln 37 − + − (orf_ABC3040) Methylobacterium populi Met31 + − − ZP_02200540 (orf_Mpop1292) ² Bacillus subtilis NP_388092 Gln41 + + + (α/β-fatty acid hydroxylase, P450_(Bsβ)) Sphingomonaspaucimobilis Gln 36 − + − BAA22987 (α-fatty acid hydroxylase,P450_(Spα)) ¹ in-vitro experiments with lysate, not with purifiedprotein, difficult to detect hydroxyl fatty acids ² poorly expressed inE. coli, activity was only detected in-vitro ³ Relative to the positionin ORF880 ⁴ with hexadecanoic acid as substrate (in-vitro and in-vivo)hyFA = hydroxy fatty acid

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A recombinant host cell engineered to produce an olefin, comprising a polynucleotide sequence encoding a polypeptide having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 7, wherein the polypeptide has fatty acid decarboxylase activity.
 2. The recombinant host cell of claim 1, wherein the polynucleotide sequence is endogenous to the microorganism.
 3. The recombinant host cell of claim 1, wherein the polynucleotide sequence is heterologous to the microorganism.
 4. The recombinant host cell of claim 1, wherein the recombinant host cell expresses an increased level of the polypeptide having fatty acid decarboxylase activity relative to a corresponding wild-type host cell.
 5. The recombinant host cell of claim 1, wherein a gene encoding an acyl-CoA synthase is attenuated or deleted in the host cell and wherein the host cell expresses a reduced level of an acyl-CoA synthase relative to a corresponding wild type microorganism.
 6. The recombinant host cell of claim 5, wherein the acyl-CoA synthase gene is fadD.
 7. The recombinant host cell of claim 4, wherein the recombinant host cell is engineered to express, overexpress, or attenuate expression of a thioesterase.
 8. A cell culture comprising the recombinant host cell of claim 4, wherein an olefin is produced in the cell culture when the recombinant host cell is cultured in medium containing a substrate under conditions effective to express the polypeptide having fatty acid decarboxylase activity.
 9. A cell culture comprising the recombinant host cell of claim 7, wherein an olefin is produced in the cell culture when the recombinant host cell is cultured in medium containing a substrate under conditions effective to express the polypeptide having fatty acid decarboxylase activity.
 10. The cell culture of claim 8, wherein the olefin is a terminal olefin.
 11. The cell culture of claim 8, wherein the olefin is secreted by the host cell.
 12. The cell culture of claim 10, wherein the terminal olefin is selected from the group consisting of a C₅-C₂₅ terminal olefin, a C₁₃-C₂₁ terminal olefin, nonadecene, methylnonadecene, heptadecene, methylheptadecene, and pentadecene, an unsaturated terminal olefin, a monounsaturated terminal olefin, a straight chain terminal olefin, a branched chain terminal olefin, and a terminal olefin comprising a cyclic moiety.
 13. The cell culture of claim 8, wherein the substrate is a carbon source.
 14. The cell culture of claim 13, wherein the carbon source is a fatty acid.
 15. The cell culture of claim 13, wherein the carbon source is a carbohydrate.
 16. The cell culture of claim 14, wherein the fatty acid is selected from the group consisting of a C₆-C₂₆ fatty acid, a C₁₄-C₂₂ fatty acid, tetradecanoic acid, hexadecanoic acid, octadecanoic acid, behenic acid, methyl eicosanoic acid, eicosanoic acid, methyl octadecanoic acid, stearic acid, and palmitic acid, an unsaturated fatty acid, a monounsaturated fatty acid, a saturated fatty acid, a straight chain fatty acid, and a cyclic moiety.
 17. A method of producing an olefin comprising culturing the host cell of claim 1, under conditions sufficient to allow expression of the fatty acid decarboxylase polypeptide.
 18. The method of claim 17, further comprising culturing the host cell in the presence of at least one biological substrate for the polypeptide.
 19. The method of claim 18, further comprising isolating the olefin from the culture medium.
 20. An olefin produced by the method of claim 19, wherein the olefin has a δ¹³C selected from the group consisting of about −15.4 or greater, about −15.4 to about −10.9, and about −13.92 to about −13.84.
 21. An olefin produced by the method of claim 19, wherein the olefin has an f_(M) ¹⁴C selected from the group consisting of at least about 1.003, at least about 1.01, at least about 1.5, and about 1.111 to about 1.124. 